Methods and Compositions for Treatment of Autism

ABSTRACT

Disclosed herein are methods and compositions for treating autism. Disclosed herein are methods and compositions for treating an autism spectrum disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 13/665,679, which claims priority to U.S.Provisional Patent Application No. 61/558,486 filed Nov. 11, 2011, toU.S. Provisional Patent Application No. 61/558,094 filed Nov. 10, 2011,and to U.S. Provisional Patent Application No. 61/553,509 filed Oct. 31,2011, which applications are incorporated herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under GM077456, HD10981and HD20961 awarded by Johns Hopkins McKusick-Nathans Institute ofGenetic Medicine and Public Health Service. The government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Oct. 10, 2014 as a text file named“36406_(—)0005U2_Sequence_Listing.txt,” created on Oct. 10, 2014, andhaving a size of 12,643 bytes is hereby incorporated by referencepursuant to 37 C.F.R. §1.52(e)(5).

BACKGROUND

Autism is currently one of five disorders that fall under the umbrellaof Pervasive Developmental Disorders (PDD), a category of neurologicaldisorders characterized by severe and pervasive impairment in severalareas of development. Autism is a complex developmental disability thattypically appears during the first two years of life and affects thefunctioning of the brain, impacting development of social interactionand communication skills. Both children and adults on the autismspectrum typically show difficulties in verbal and non-verbalcommunication, social interactions, and leisure or play activities.Autism knows no racial, ethnic, or social boundaries, and can affect anyfamily and any child.

Autism statistics from the U.S. Centers for Disease Control andPrevention (CDC) identify around 1 in 88 American children as on theautism spectrum—a ten-fold increase in prevalence in 40 years. Carefulresearch shows that this increase is only partly explained by improveddiagnosis and awareness. Studies also show that autism is four to fivetimes more common among boys than girls. An estimated 1 out of 54 boysand 1 in 252 girls are diagnosed with autism in the United States.

Despite progress in genetic research in autism spectrum disorders (ASD),direct treatment of underlying abnormal cellular mechanisms in ASD isnot yet possible. Genetic associations correlate with at most only ˜20%of ASD patients. This indicates considerable clinical heterogeneity,which likely results from multiple cellular mechanisms. Several clinicaland laboratory findings in ASD indicate that different types of cellulardysfunction, including neuro-inflammation (Vargas et al., 2005),oxidative stress (James et al., 2009), and mitochondrial abnormalities(Weissman et al., 2008), and abnormal synaptic plasticity andconnectivity (Weng et al., 2010), involve a number of related,interacting metabolic pathways.

The medications and treatments currently used to treat autism and autismspectrum disorders are symptomatic; there is no evidence that thesemedications either improve core features (e.g., social responsiveness)or affect the neurodevelopmental trajectory. Thus, despite intensiveefforts, no effective methods for treatment or prevention of autism areavailable.

What is needed is a mechanism-based strategy that targets the intrinsiccellular stress response and modulates the metabolic defects thatcontribute to the symptomotology of autism and autism spectrumdisorders.

SUMMARY

Disclosed herein is a method of treating autism, comprising,administering to a subject diagnosed with autism an effective amount ofa composition comprising a compound that induces a general cellularstress response in at least one cell of the subject; and allowing thesubject's treated or affected cells to return to cellular homeostasisthat existed prior to administering the compound. A composition maycomprise a pharmaceutical composition, a natural product composition, amedical food, a nutritional supplement, or a composition comprisingexcipients, diluents, enzymes, cofactors, and delivery vehicleadditives. The treatment of subjects with compounds disclosed hereincomprise a return, by treated and/or affected cells, to a statesubstantially similar to the state of the treated cells prior totreatment, herein referred to as a return to cellular homeostasis. Inthe treated subject as a whole, the symptoms of and state of thesubject's autism or autism spectrum disorder is reduced by such atreatment, so that the subject has lessened, or fewer attributes of,autism or autism spectrum disorder.

Disclosed herein is a method of treating autism, comprising,administering to at least one cell of a subject diagnosed with autism aneffective amount of a composition comprising a compound that induces ageneral cellular stress response in the at least one cell of thesubject; and allowing the cell to return to homeostasis that existedprior to administering the compound. A composition may comprise apharmaceutical composition, a natural product composition, a medicalfood, a nutritional supplement, or a composition comprising excipients,diluents, enzymes, cofactors, and delivery vehicle additives.

Disclosed herein is a method of treating autism, comprising,administering to a subject diagnosed with autism an effective amount ofa composition that modulates measurable effects of behavioral symptoms.A composition may comprise a pharmaceutical composition, a naturalproduct composition, a medical food, a nutritional supplement, or acomposition comprising excipients, diluents, enzymes, cofactors, anddelivery vehicle additives.

Disclosed herein is a method of treating autism, comprising,administering to a subject diagnosed with autism an effective amount ofa composition that modulates social responsiveness of the subjecttreated. A composition may comprise a pharmaceutical composition, anatural product composition, a medical food, a nutritional supplement,or a composition comprising excipients, diluents, enzymes, cofactors,and delivery vehicle additives.

Disclosed herein is a method of treating one or more autism spectrumdisorders, comprising, administering to a subject diagnosed with one ormore autism spectrum disorders an effective amount of a compositioncomprising a compound that induces a general cellular stress response inat least one cell of the person. A composition may comprise apharmaceutical composition, a natural product composition, a medicalfood, a nutritional supplement, or a composition comprising excipients,diluents, enzymes, cofactors, and delivery vehicle additives.

Disclosed herein is a method of treating one or more autism spectrumdisorders, comprising, administering to a subject diagnosed with one ormore autism spectrum disorders an effective amount of a composition thatmodulates measurable effects of behavioral symptoms. A composition maycomprise a pharmaceutical composition, a natural product composition, amedical food, a nutritional supplement, or a composition comprisingexcipients, diluents, enzymes, cofactors, and delivery vehicleadditives.

Disclosed herein is a method of treating one or more autism spectrumdisorders, comprising, administering to a subject diagnosed with one ormore autism spectrum disorders an effective amount of a composition thatmodulates social responsiveness of the subject treated. A compositionmay comprise a pharmaceutical composition, a natural productcomposition, a medical food, a nutritional supplement, or a compositioncomprising excipients, diluents, enzymes, cofactors, and deliveryvehicle additives.

Disclosed herein is a method for determining effectiveness of a compoundin treating autism or autism related disorder, comprising, administeringto a first cell an effective amount of a compound to be tested,comparing the response of the first cell with the response of anidentical cell that was treated with a compound that induces ageneralized stress response, and determining whether or not the testedcompound induces a general cellular stress response in cells.

Disclosed herein are medical foods used to treat autism or one or moreautism spectrum disorders.

Disclosed herein are dietary or nutritional supplements used to treatautism or one or more autism spectrum disorders.

Disclosed herein are compounds derived from natural or synthetic sourceswhich may be used in a composition to treat autism or one or more autismspectrum disorders.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying Figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1 (A-E) shows that the beneficial responses in XALD and K562 cellsinvolves pharmacological induction of mitochondrial function

FIG. 2 (A-F) shows that the mitochondrial biogenesis induced by 4PBA,HU, TSA, or SFN is JNK-dependent.

FIG. 3 (A-B) shows that the heat shock response is induced by 4PBA, HU,TSA, or SFN treatment.

FIG. 4 (A-C) shows that the unfolded protein response is activated bytreatment with 4PBA, HU, TSA, or SFN.

FIG. 5 (A-D) shows that autophagy and the antioxidant response areinduced by treatment with 4PBA, HU, TSA, or SFN.

FIG. 6 (A-B) shows that HU and SFN do not inhibit class I and class IIhistone deacetylase activities.

FIG. 7 shows the induction of mitochondrial biogenesis by 4BPA, HU, TSA,and SFN in spinal muscular atrophy fibroblasts.

FIG. 8 (A-D) shows the induction of FL-SMN and SMN expression by SFN isdependent upon the JNK pathway, autophagy, mitochondrial biogenesis, andSIRT1 activity.

FIG. 9 shows that the biochemical SIRT1 activity is not required for theinduction of the stress proteome by 4PBA, HU, TSA, and SFN.

DESCRIPTION A. Autism and Autism Spectrum Disorders

Autism spectrum disorder (ASD) and autism are both general terms for agroup of complex disorders of brain development. These disorders arecharacterized, in varying degrees, by difficulties in socialinteraction, verbal and nonverbal communication and repetitivebehaviors. These disorders include, but are not limited to, autisticdisorder, Rett syndrome, childhood disintegrative disorder, pervasivedevelopmental disorder—not otherwise specified (PDD-NOS) and Aspergersyndrome. ASD can be associated with intellectual disability,difficulties in motor coordination and attention and physical healthissues such as sleep and gastrointestinal disturbances. Some personswith ASD excel in visual skills, music, math and art. (See, e.g.,Wiggins et al. 2011, which is incorporated herein in its entirety byreference, and which shows that adolescents with ASD have weakerconnectivity between the posterior hub of the default network and theright superior frontal gyms). Though not wishing to be bound by anyparticular theory, it is believed that autism spectrum disorders mayresult from a variety of non-lethal genetic disorders and epigeneticeffects that affect related metabolic pathways.

such as self-organizing maps (SOM), to create a reference for eachparticipant to calculate connectivity. We used individualizedresting-state clusters identified by an SOM algorithm to corroborateprevious findings of weaker posterior-anterior connectivity in the ASDgroup and examine age-related changes in the ASD and control groups.Thirty-nine adolescents with ASD and 41 controls underwent a 10-minute,eyes-open, resting-state functional MRI scan. Additionally, controlshave larger increases in connectivity with age compared to the ASDgroup. These findings indicate that SOM is a complementary method forcalculating connectivity in a clinical population. Additionally,adolescents with ASD have a different developmental trajectory of thedefault network compared to controls.

Behaviors in 38% (Miles 2010) to 83% (Curran et al., 2007) of autisticchildren transiently improved during episodes of fever. Improvementswere most notable in reduced stereotypic behaviors and inappropriatespeech. Improvements were not related to the degree of fever or severityof illness (Curran et al., 2007, which is incorporated in its entiretyby this reference). Fever stimulates the HSP, which are important formultiple cellular processes in the CNS, including synaptic transmission.(Stetler et al., 2010).

The cellular stress proteome is a complex of interacting proteinsinvolved in pathways that respond to various stressors, such as fever,irradiation and hypoxia, and functions to re-establish homeostasis.Stress response pathways are not stressor specific and include organellebiogenesis. MAPK signaling, antioxidant production, heat shock proteins,unfolded protein responses, and autophagy (Stetler et al., 2010).

B. Compositions

i) Glucosinolates

The present invention comprises methods and compositions comprisingglucosinolates for the treatment of autism and autism spectrumdisorders. Though not wishing to be bound by any particular theory, itis currently believed that a benefit of crucifer plants is their contentof isothiocyanates and their precursor molecules, glucosinolates.Glucosinolates are converted to isothiocyanates by the enzymes such asthioglucosides, for example, myrosinase. Generally, in plant cells,myrosinase and glucosinolates are separated in the cell and if the cellis damaged, such as by insect predation, with loss ofcompartmentalization, myrosinase or other similarly acting enzymes comesinto contact with glucosinolates, which are then converted toisothiocyanates. Myrosinase, EC 3.2.1.147, CAS number 9025-38-1 is knownto those of skill in the art, as are similarly acting enzymes thatconvert precursor molecules, such as glucosinolates, to more activecompounds, such as isothiocyanates, such as sulforaphane.

The present invention comprises methods and compositions comprising oneor more enzymes, and/or one or more types of enzymes, and optionallyco-factors or other enzymes in the metabolic pathway, for the treatmentof autism and autism spectrum disorders. Enzymes contemplated by thepresent invention, referred to herein as enzymes of the presentinvention, comprise, but are not limited to, myrosinase,thioglucosidases, glutathione transferases, NAD(P)H:quinone reductase(QR) and glucuronosyltransferases, which have similar activities or arein related pathways. For example, as known in the art, in the presenceof water, myrosinase cleaves the glucose group from a glucosinolate. Theremaining molecule then converts to a thiocyanate, an isothiocyanate ora nitrile; these are the active substances that serve as defense for theplant. The hydrolysis of glucosinolates by myrosinase or other enzymesof the present invention or similarly acting enzymes can yield a varietyof products, depending on various physiological conditions such as pHand the presence of certain cofactors. Reactions have been observed toshare the initial steps. First, the β-thioglucoside linkage is cleavedby myrosinase, releasing D-glucose. The resulting aglycone undergoes aspontaneous Lossen-like rearrangement, releasing a sulfate. The laststep in the mechanism is subject to the greatest variety depending onthe physiological conditions under which the reaction takes place. Atneutral pH, the primary product is the isothiocyanate. Under acidicconditions (pH<3), and in the presence of ferrous ions orepithiospecifer proteins, the formation of nitriles is favored instead.

Methods for the extraction of natural products as sources for compoundssuch as sulforaphane, include methods for extraction from plant sourcesin contrast to those produced by chemical synthetic methods, such asfrom plant sources such as cruciferous vegetables include, but are notlimited to homogenization of the vegetables in cold water,lyophilization, extraction of the resultant powder with acetonitrile,filtration and evaporative concentration. Other methods for extractionof compounds from plants are known in the art and are contemplated bythe present invention, and may comprise, for example, extractions ofseeds and sprouts to produce compounds of the present invention, such astaught by U.S. Pat. No. 5,725,895, which is herein incorporated in itsentirety. Known methods for extracting natural products, particularlyfrom cruciferous plants, comprise extraction methods comprising boilingwater extraction of desired compounds.

Though not wishing to be bound by any particular theory, it is currentlybelieved that glucosinolates are a precursor molecule without activitythat is then converted by enzymes into an active form, for example anisothiocynate such as sulforaphane (which may be referred to herein as amore active compound because the compound is more active in particularassays than the activity of its precursor molecule. Compositions of thepresent invention comprise one or more precursor compounds, such asglucosinolates, and/or may comprise more active molecules such asproducts made by enzymatic activity on glucosinolates, for examplesulforaphane, or both one or more precursor compounds and one or moreactive compounds, and further may optionally comprise one or moreenzymes and/or co-factors of such enzymes that use the precursorcompound or the more active compound as a substrate. Glucosinolates infoods are converted at least partially to isothiocyanates in humans, by,it is currently believed, microorganisms of the gut. For example, acomposition of the present invention may comprise one or more precursorcompounds, such as glucosinolate, for the treatment of autism and autismspectrum disorders. A composition may further comprise one or moreenzymes for which the compound provided in the composition is asubstrate molecule of the one or more enzymes. The composition may beprovided in a unitary delivery vehicle or may be provided in two or moredelivery vehicles which may be provided simultaneously, sequentially, orin other administrative methods.

ii) Cruciferous Plant Sources

Plant sources suitable for use in the methods and compositions disclosedherein may be any portion of a cruciferous plant, including, but notlimited to cells, seeds, sprouts, leaves, stalks, roots, flowers andother plant structures. Plant sources contemplated by the presentinvention comprise, but are not limited to, plants from the familyCruciferae, such as Brassiceae, and including Brassicinae. For example,the plant source may be Brassica oleracea selected from the group ofvarieties of acephala (kale, collards, wild cabbage, curly kale),medullosa (marrowstem kale), ramosa (thousand head kale), alboglabra(Chinese kale), botrytis (cauliflower, sprouting broccoli), costata(Portuguese kale), gemmifera (Brussels sprouts), gongylodes (kohlrabi),italica (broccoli), palmifolia (Jersey kale), sabauda (savoy cabbage),sabellica (collards), and selensia (borecole), among others.

Useful broccoli cultivars to be used in the method and compositionsdisclosed herein are Saga, DeCicco, Everest, Emerald City, Packman,Corvet, Dandy Early, Emperor, Mariner, Green Comet, Green Valiant,Arcadia, Calabrese Caravel, Chancellor, Citation, Cruiser, Early PurpleSprouting Red Arrow, Eureka, Excelsior, Galleon, Ging a, Goliath, GreenDuke, Greenbelt, Italian Sprouting, Late Purple Sprouting, Late WinterSprouting White Star, Legend, Leprechaun, Marathon, Mariner, Minaret(Romanesco), Paragon, Patriot, Premium Crop, Rapine (Spring Raab),Rosalind, Salade (Fall Raab), Samurai, Shogun, Sprinter, Sultan, Taiko,and Trixie. However, many other broccoli cultivars are suitable.

Useful cauliflower cultivars to be used in the method and compositionsdisclosed herein are Alverda, Amazing, Andes, Burgundy Queen, CandidCharm, Cashmere, Christmas White, Dominant, Elby, Extra Early Snowball,Fremont, Incline, Milkyway Minuteman, Rushmore, S-207, Serrano, SierraNevada, Siria, Snow Crown, Snow Flake, Snow Grace, Snowbred, Solide,Taipan, Violet Queen, White Baron, White Bishop, White Contessa, WhiteCorona, White Dove, White Flash, White Fox, White Knight, White Light,White Queen, White Rock, White Sails, White Summer, White Top, Yukon.However, many other cauliflower cultivars are suitable.

iii) Sulforaphane

A composition of the present invention comprises sulforaphane, anorganosulfur compound (1-isothiocyanato-4R-(methylsulfinyl)butane),derivatives known in the art, such as dithiocarbamate derivatives andothers disclosed herein, and analogs known in the art and/or disclosedherein. Sulforaphane is a hormetic drug that, though not wishing to bebound by any particular theory, is thought to induce a general “cellprotective” response. Sulforaphane is an active component of many plantsand for example, may be extracted from broccoli sprouts, or may be madeby chemical synthetic methods. Sulforaphane may be obtained fromlyophilized, freeze dried extracts of 3-day-old broccoli sprouts.Broccoli sprouts are widely consumed all over the world by a very largenumber of individuals, without any reports of adverse effects. Humanresearch studies have also not shown any significant adverse effects byadministration of sulforaphane.

Sulforaphane crosses the blood brain barrier. Studies have demonstratedbioavailability of sulforaphane to the brain, peripheral nervous system,and nerve cells. Studies in various strains of mice and rats havedemonstrated the accumulation of sulforaphane (and its dithiocarbamatemetabolites) in the brain after various routes of administration (Zhaoet al., 2005 and Clarke et al., 2011, both of which is incorporated intheir entirety by this reference).

Though not wishing to be bound by any particular theory, it is believedthat sulfurophane may provide protection against oxidative andinflammatory stress, such as disturbances of systems that protect cellsagainst oxidative damage, heat shock, and disturbances caused by proteinmisfolding. These protective mechanisms are thought to be mediated bythe transcription factor Nrf2 which controls expression of genes of thehuman genome via the Keap1/Nrf2/ARE regulatory system. This system maybe upregulated in many tissues including the brain by sulforaphane.(See, e.g., Baird et al., 2011, which is incorporated in its entirety bythis reference).

Data has shown that heat shock protein and ubiquitin 26s proteasomesubunits were induced in mice after treatment with sulforaphane (Hu etal., 2008). Heat shock factor 1 (HSF1) and expression of HSP 27 wereamplified by sulforaphane. In HeLa cells, sulforaphane upregulates HSP70and HSP90. Others have found that the Keap1-Nrf2 Pathway may beinvolved. (Baird et al., 2011).

The art is familiar with sulforaphane analogs, which include, but arenot limited to, the following: 6-isothiocyanato-2-hexanone,exo-2-acetyl-6-isothiocyanatonorbornane,exo-2-isothiocyanato-6-methylsulfonylnorbornane,6-isothiocyanato-2-hexanol, 1-isothiocyanato-4-dimethylphosphonylbutane,exo-2-(1′-hydroxyethyl)-5-isothiocyanatonorbornane,exo-2-acetyl-5-isothiocyanatonorbornane,1-isothiocyanato-5-methylsulfonylpentane,cis-3-(methylsulfonyl)cyclohexylmethylisothiocyanante andtrans-3-(methylsulfonyl)cyclohexylmethylisothiocyanante.

iv) Compounds of the Present Invention

A composition of the present invention may comprise one or morecompounds disclosed herein, for example, histone deacetylase inhibitors,Class I and class II histone deacetylase inhibitors, and compounds thatare not histone deacetylase inhibitors, such as hydroxyurea,sulforaphane and/or its derivatives and analogs. A composition of thepresent invention may comprise a compound that modulates one or moreaspects of the general cellular stress response of a cell. It iscurrently believed that one or more pathways may be involved inmodulating the general cellular stress response of a cell, and thepresent invention contemplates compounds, including, but not limited tothose disclosed herein, that up-regulate the general cellular stressresponse of a cell, as described herein and as is commonly understood inthe art.

For example, in an aspect, a composition comprises 4-phenylbutyrate. Inan aspect, a composition comprises sodium butyrate. In an aspect, acomposition comprises hydroxyurea. In an aspect, a composition comprisessulforaphane. In an aspect, a composition comprises derivatives ofsulforaphane. In an aspect, a composition comprises analogs ofsulforaphane. In an aspect, a composition comprises trichostatin A. Inan aspect, a composition comprises a combination of compounds disclosedherein, for example a composition comprising one or more of hydroxyurea,sulforaphane, 4-phenylbutyrate, sodium butyrate, and/or trichostatin A.For example, a composition comprises a combination comprisingphenylbutyrate and sodium butyrate. In an aspect, a compositioncomprises a combination comprising phenylbutyrate and trichostatin A. Inan aspect, a composition comprises a combination comprising sodiumbutyrate and trichostatin A. In an aspect, a composition comprises acombination comprising hydroxyurea, phenylbutyrate, sodium butyrate. Inan aspect, a composition comprises a combination comprising hydroxyureaand sulforaphane. In an aspect, a composition comprises a combinationcomprising hydroxyurea and sulforaphane derivatives or analogs, as knownand/or disclosed herein. In an aspect, a composition comprises acombination comprising hydroxyurea, sulforaphane, phenylbutyrate, sodiumbutyrate, and trichostatin A. In an aspect, a composition comprises acombination comprising sulforaphane, phenylbutyrate, sodium butyrate,and trichostatin A. In an aspect, a composition comprises a combinationcomprising sulforaphane derivatives or analogs, and one or more ofphenylbutyrate, sodium butyrate, and trichostatin A.

A composition of the present invention may comprise a compound that ispresent as a structure represented by a formula:

or a subgroup or pharmaceutically acceptable salt thereof.

v) Compounds that Modulate the General Cell Stress Response

The present invention comprises methods and compositions comprisingcompounds for the treatment of autism and autism spectrum disorders thatmodulate the general cellular stress response that modulate, forexample, transcription and expression of heat shock factors and heatshock proteins. Heat shock proteins (HSPs) are involved in sensing andrepairing DNA damage and function as molecular chaperones in manypathways. Heat shock factors (HSF) are transcriptional regulators forHSP that act as stress integrators in cells and organisms to maintainhomeostasis and are evolutionarily conserved. (Akerfelt et al., 2010).

Methods of the present invention comprise administering an effectiveamount of a compound disclosed herein, such as histone deacetylase(HDAC) inhibitors and non-HDAC inhibitors, such as hydroxyurea orsulforaphane, to modulate the general cellular stress response, such asmodulating levels and amounts of nucleic acids and peptides and proteinsassociated with the general cellular stress response, includingmitochondrial biogenesis, peroxisome proliferation, activation of thestress proteome, transcription and/or translation of genes and proteinsencoded by genes comprising heat shock and unfolded protein, genes forautophagic responses, genes for antioxidant responses, and genes for thec-jun-N-terminal kinase pathway.

A composition of the present invention that modulates the general stressresponse in a cell may comprise a compound that is present as astructure represented by a formula:

or a subgroup or pharmaceutically acceptable salt thereof.

Compositions of the present invention may comprise a compound disclosedherein that is formulated as a medical food. A composition of thepresent invention comprises a medical food comprising a compounddisclosed herein and components known to those of skill in the art forproducing medical foods. Medical foods are foods that are speciallyformulated and intended for the dietary management of a disease, such asautism and/or autism spectrum disorder, which has distinctivenutritional needs that cannot be met by normal diet alone. In an aspect,the disease, for example, is autism. In an aspect, the disease, forexample, is one or more autism spectrum disorders. As defined by the FDAin section 5(b) of the Orphan Drug Act (21 U.S.C. 360ee (b) (3)), theterm “medical food” is defined as “a food which is formulated to beconsumed or administered enterally under the supervision of a physicianand which is intended for the specific dietary management of a diseaseor condition for which distinctive nutritional requirements, based onrecognized scientific principles, are established by medicalevaluation.”

vi) Medical Foods

Medical foods are distinct from the broader category of foods forspecial dietary use and from traditional foods that bear a health claim.In order to be considered a medical food the product must, at a minimum:(i) be a food for oral ingestion or tube feeding (nasogastric tube),(ii) be labeled for the dietary management of a specific medicaldisorder, disease or condition for which there are distinctivenutritional requirements, and (iii) be intended to be used under medicalsupervision. In an aspect, the disease for which there are distinctivenutritional requirements is, for example, autism. In an aspect, thedisease for which there are distinctive nutritional requirements is, forexample, one or more autism spectrum disorders.

Disclosed herein are medical foods used to treat autism. Disclosedherein are medical foods used to treat one or more autism spectrumdisorders.

A composition of the present invention comprises a medical foodcomprising one or more compounds disclosed herein, one or more compoundsthat modulate the general cellular stress response, one or more histonedeacetylase inhibitors, Class I histone deacetylase inhibitors, and orClass II histone deacetylase inhibitors. For example, in an aspect, themedical food comprises 4-phenylbutyrate. In an aspect, the medical foodcomprises sodium butyrate. In an aspect, the medical food comprisestrichostatin A. In an aspect, the medical food comprises a combinationof 4-phenylbutyrate, sodium butyrate, and/or trichostatin A. Forexample, the medical food comprises a combination comprisingphenylbutyrate and sodium butyrate. In an aspect, the medical foodcomprises a combination comprising phenylbutyrate and trichostatin A. Inan aspect, the medical food comprises a combination comprising sodiumbutyrate and trichostatin A. In an aspect, the medical food comprises acombination comprising phenylbutyrate, sodium butyrate, and trichostatinA. In an aspect, the medical food comprises hydroxyurea. In an aspect,the medical food comprises one or more Class II histone deacetylaseinhibitors. In an aspect, the medical food comprises sulforaphane or asulforaphane analog. In an aspect, the medical food comprises asulforaphane dithocarbamate metabolite.

In an aspect, the medical food comprises at least one of a histonedeacetylase inhibitor, a Class I histone deacetylase inhibitor, a ClassII histone deacetylase inhibitor, 4-phenylbutyrate, trichostatin A,hydroxyurea, sulforaphane or a sulforaphane analog, a sulforaphanederivative, or a sulforaphane dithocarbamate metabolite. In an aspect,the medical food comprises a combination of a histone deacetylaseinhibitor, a Class I histone deacetylase inhibitor, a Class II histonedeacetylase inhibitor, 4-phenylbutyrate, trichostatin A, hydroxyurea,sulforaphane or a sulforaphane analog, a sulforaphane derivative, or asulforaphane dithocarbamate metabolite. In an aspect, the medical foodcomprises a combination of a histone deacetylase inhibitor, such as forexample, a Class I histone deacetylase inhibitor or a Class II histonedeacetylase inhibitor, and one or more of the following:4-phenylbutyrate, trichostatin A, hydroxyurea, sulforaphane, asulforaphane derivative, or a sulforaphane analog, or a sulforaphanedithocarbamate metabolite. In an aspect, the medical food comprises acombination of 4-phenylbutyrate, trichostatin A, hydroxyurea, andsulforaphane or a sulforaphane analog.

In an aspect, the medical food comprises one or more cruciferous seedsor sprouts, or the extracts from one or more cruciferous sprouts.

In a disclosed method of treating autism or one or more autism spectrumdisorders, the medical food comprises a compound that is present as astructure represented by a formula:

or a subgroup or pharmaceutically acceptable salt thereof.

In a disclosed method of treating autism or one or more autism spectrumdisorders, a sulforaphane analog is 6-isothiocyanato-2-hexanone,exo-2-acetyl-6-isothiocyanatonorbornane,exo-2-isothiocyanato-6-methylsulfonylnorbornane,6-isothiocyanato-2-hexanol, 1-isothiocyanato-4-dimethylphosphonylbutane,exo-2-(1′-hydroxyethyl)-5-isothiocyanatonorbornane,exo-2-acetyl-5-isothiocyanatonorbornane,1-isothiocyanato-5-methylsulfonylpentane,cis-3-(methylsulfonyl)cyclohexylmethylisothiocyanante, ortrans-3-(methylsulfonyl)cyclohexylmethylisothiocyanante.

In a disclosed method of treating autism or one or more autism spectrumdisorders, the medical food comprises excipients or diluents.

In an aspect, the disclosed medical food comprises one or more compoundsthat causes (i) mitochondrial biogenesis, (2) peroxisome proliferation,(3) activation of the stress proteome, or (4) transcription and/ortranslation of genes and proteins encoded by (a) genes comprising heatshock and unfolded protein, (b) genes for autophagic responses, (c)genes for antioxidant responses, and (d) genes for the c-jun-N-terminalkinase pathway.

In an aspect, a medical food disclosed herein comprises compounds andcompositions of the present invention, and optionally comprises enzymesfor which compounds of the present invention are substrates. Medicalfood compositions may be taken by subjects in a delivery vehicleappropriate for the route of administration, such as capsules, topicalcreams, nasal sprays, injectable solutions, pastilles, sachets, and suchcompositions may be administered alone or in combination with othercomponent compositions. For example, a composition may comprisecompounds of the present invention in one delivery vehicle that isadministered concurrently or sequentially with a second compositioncomprising an enzyme composition of the present invention. An enzymecomposition as used herein may comprise one or more disclosed enzymesfor altering precursor molecules to result in more active compounds, andmay comprise co-factors, co-enzymes, and/or other enzymes in aconversion pathway.

vii) Dietary Supplements

The present invention comprises methods and compositions comprisingcompounds disclosed herein for the treatment of autism and autismspectrum disorders that are formulated as dietary or nutritionalsupplements. A dietary supplement, also known as food supplement ornutritional supplement, is a preparation intended to supplement the dietand provide compounds, such as those disclosed herein or such asvitamins, minerals, fiber, fatty acids, or amino acids, that may bemissing from normal dietary sources or may not be consumed in sufficientquantities in a person's diet. Some countries define dietary supplementsas foods, while in others they are defined as drugs or natural healthproducts. A dietary supplement may be provided in delivery vehiclessuitable for administration, or may be added to foods, liquids, solids,drinks, water, or other ingestible or nutritious compositions thatsubjects can eat or drink.

Disclosed herein are dietary supplements used to treat autism. Disclosedherein are dietary supplements used to treat one or more autism spectrumdisorders.

A composition of the present invention comprises a dietary supplementcomprising one or more compounds disclosed herein, one or more compoundsthat modulate the general cellular stress response, one or more histonedeacetylase inhibitors, Class I histone deacetylase inhibitors, and orClass II histone deacetylase inhibitors. In an aspect, a dietarysupplement comprises one or more Class I histone deacetylase inhibitors.For example, in an aspect, a dietary supplement comprises4-phenylbutyrate. In an aspect, a dietary supplement comprises sodiumbutyrate. In an aspect, a dietary supplement comprises trichostatin A.In an aspect, a dietary supplement comprises a combination of4-phenylbutyrate, sodium butyrate, and/or trichostatin A. For example, adietary supplement comprises a combination comprising phenylbutyrate andsodium butyrate. In an aspect, the dietary supplement comprises acombination comprising phenylbutyrate and trichostatin A. In an aspect,the dietary supplement comprises a combination comprising sodiumbutyrate and trichostatin A. In an aspect, the dietary supplementcomprises a combination comprising phenylbutyrate, sodium butyrate, andtrichostatin A. In an aspect, the dietary supplement compriseshydroxyurea. In an aspect, the dietary supplement comprises one or moreClass II histone deacetylase inhibitors. In an aspect, the dietarysupplement comprises sulforaphane or a sulforaphane analog orderivative. In an aspect, the dietary supplement comprises asulforaphane dithocarbamate metabolite.

In an aspect, the dietary supplement comprises at least one of a histonedeacetylase inhibitor, a Class I histone deacetylase inhibitor, a ClassII histone deacetylase inhibitor, 4-phenylbutyrate, trichostatin A,hydroxyurea, sulforaphane or a sulforaphane analog, or a sulforaphanedithocarbamate metabolite. In an aspect, the dietary supplementcomprises a combination of a histone deacetylase inhibitor, a Class Ihistone deacetylase inhibitor, a Class II histone deacetylase inhibitor,4-phenylbutyrate, trichostatin A, hydroxyurea, sulforaphane or asulforaphane analog, or a sulforaphane dithocarbamate metabolite. In anaspect, the dietary supplement comprises a combination of a histonedeacetylase inhibitor, such as for example, a Class I histonedeacetylase inhibitor or a Class II histone deacetylase inhibitor, andone or more of the following: 4-phenylbutyrate, trichostatin A,hydroxyurea, sulforaphane or a sulforaphane analog, or a sulforaphanedithocarbamate metabolite. In an aspect, the dietary supplementcomprises a combination of 4-phenylbutyrate, trichostatin A,hydroxyurea, and sulforaphane or a sulforaphane analog.

A composition of the present invention comprises a dietary supplementcomprising a compound that is present as a structure represented by aformula:

or a subgroup or a pharmaceutically acceptable salt thereof.

In a disclosed method of treating autism or one or more autism spectrumdisorders, the dietary supplement comprises excipients or diluents, orother components routinely used in formulating a dietary supplement.

In an aspect, the disclosed dietary supplement comprises one or morecruciferous sprouts, or the extracts from one or more cruciferous plantsources.

In an aspect, a disclosed dietary supplement comprises one or morecompounds that causes (i) mitochondrial biogenesis, (2) peroxisomeproliferation, (3) activation of the stress proteome, or (4)transcription and/or translation of genes and proteins encoded by (a)genes comprising heat shock and unfolded protein, (b) genes forautophagic responses, (c) genes for antioxidant responses, and (d) genesfor the c-jun-N-terminal kinase pathway.

In an aspect, the dietary supplements disclosed herein comprisecompounds and compositions of the present invention, and optionallycomprises enzyme compositions that alter compounds of the presentinvention. Dietary supplements compositions may be taken by subjects ina delivery vehicle appropriate for the route administration, such ascapsules, topical creams, nasal sprays, injectable solutions, pastilles,sachets, and such compositions may be administered alone or incombination with other component compositions. For example, in a method,one composition may comprise compounds of the present invention in onedelivery vehicle that is administered concurrently or sequentially witha second composition comprising an enzyme composition of the presentinvention.

B. Methods of Treating Autism

Disclosed herein are methods of treating autism.

Disclosed herein is a method of treating autism comprising administeringto a subject diagnosed with autism an effective amount of apharmaceutical composition, wherein the composition comprises a compoundthat induces a general cellular stress response in at least one cell ofthe subject; and allowing the subject to return to homeostasis thatexisted prior to administering the compound.

Disclosed herein is method of treating autism comprising administeringto at least one cell of a subject diagnosed with autism an effectiveamount of a pharmaceutical composition comprising a compound thatinduces a general cellular stress response in the at least one cell ofthe subject; and allowing the cell to return to homeostasis that existedprior to administering the compound.

Disclosed herein is a method of treating autism comprising administeringto a subject diagnosed with autism an effective amount of apharmaceutical composition that modulates measurable effects ofbehavioral symptoms.

Disclosed herein is a method of treating autism comprising administeringto a subject diagnosed with autism an effective amount of apharmaceutical composition that modulates social responsiveness of thesubject treated. The person skilled in the art is familiar with thesocial responsiveness of a subject with autism or autism spectrumdisorder as discussed in Constantino et al., 2003 (J Autism DevelDisorders, 33: 427-433, which discusses the Social Responsiveness Scaleor SRS—a well-validated measure of autistic traits).

In a disclosed method of treating autism, the subject is male or female.In an aspect, the subject does not denote a particular age or sex. Thus,adult and newborn subjects, as well as fetuses, whether male or female,are intended to be covered. In an aspect, the subject is a mammal.

In a disclosed method of treating autism, improvements in behavioralsymptoms comprise one or more of the following: a decrease in (i)irritability, (ii) hyperactivity, (iii) stereotypy, and/or (iv)inappropriate speech. In an aspect, improvements in behavioral symptomsinclude comprise a decrease in (i) irritability, (ii) hyperactivity,(iii) stereotypy, and (iv) inappropriate speech. In an aspect,improvements in behavioral symptoms comprise a combination of two ormore of a decrease in (i) irritability, (ii) hyperactivity, (iii)stereotypy, and/or (iv) inappropriate speech. For example, in an aspect,improvements in behavioral symptoms comprise a decrease in irritabilityand hyperactivity. For example, in an aspect, improvements in behavioralsymptoms comprise a decrease in irritability and stereotypy. Forexample, in an aspect, improvements in behavioral symptoms comprise adecrease in irritability and inappropriate speech. For example, in anaspect, improvements in behavioral symptoms comprise a decrease inhyperactivity and inappropriate speech. For example, in an aspect,improvements in behavioral symptoms comprise a decrease in stereotypyand inappropriate speech. For example, in an aspect, improvements inbehavioral symptoms comprise a decrease in irritability, hyperactivity,and stereotypy. For example, in an aspect, improvements in behavioralsymptoms comprise a decrease in irritability, hyperactivity, andinappropriate speech. For example, in an aspect, improvements inbehavioral symptoms comprise a decrease in irritability, stereotypy, andinappropriate speech. For example, in an aspect, improvements inbehavioral symptoms comprise a decrease in hyperactivity, stereotypy,and inappropriate speech. As known to the person skilled in the art,behavioral symptoms in autism and autism spectrum disorders andimprovements thereof are discussed in Aman et al., 1985.

In a disclosed method of treating autism, in at least one cell of thesubject, the stress proteome is stimulated. In a disclosed method oftreating autism, in at least one cell of the subject, increased nitrousoxide production is measured. In a disclosed method of treating autism,in at least one cell of the subject, stress-sensing organelles areincreased from an amount prior to the administration of the composition.A stress-sensing organelle can be a mitrochondrion or a peroxisome. Inan aspect, a stress-sensing organelle is a mitochondrion. In an aspect,a stress-sensing organelle is a peroxisome.

In a disclosed method of treating autism, a general cellular stressresponse of a disclosed method of treating autism comprises at least oneof the following: (1) mitochondrial biogenesis, (2) peroxisomeproliferation, (3) activation of the stress proteome, or (4)transcription and/or translation of genes and proteins encoded by (a)genes comprising heat shock and unfolded protein, (b) genes forautophagic responses, (c) genes for antioxidant responses, and (d) genesfor the c-jun-N-terminal kinase pathway. In an aspect, a generalcellular stress response comprises more than one of the following: (1)mitochondrial biogenesis, (2) peroxisome proliferation, (3) activationof the stress proteome, or (4) transcription and/or translation of genesand proteins encoded by (a) genes comprising heat shock and unfoldedprotein, (b) genes for autophagic responses, (c) genes for antioxidantresponses, and (d) genes for the c-jun-N-terminal kinase pathway.

For example, in an aspect, a combination comprises mitochondrialbiogenesis and peroxisome proliferation. In an aspect, a combinationcomprises mitochondrial biogenesis and activation of the stressproteome. In an aspect, a combination comprises mitochondrial biogenesisand transcription and/or translation of genes and proteins encoded by(a) genes comprising heat shock and unfolded protein, (b) genes forautophagic responses, (c) genes for antioxidant responses, and (d) genesfor the c-jun-N-terminal kinase pathway. In an aspect, a combinationcomprises peroxisome proliferation and activation of the stressproteome.

In an aspect, a combination comprises peroxisome proliferation andtranscription and/or translation of genes and proteins encoded by (a)genes comprising heat shock and unfolded protein, (b) genes forautophagic responses, (c) genes for antioxidant responses, and (d) genesfor the c-jun-N-terminal kinase pathway. In an aspect, a combinationcomprises activation of the stress proteome and transcription and/ortranslation of genes and proteins encoded by (a) genes comprising heatshock and unfolded protein, (b) genes for autophagic responses, (c)genes for antioxidant responses, and (d) genes for the c-jun-N-terminalkinase pathway. In an aspect, the combination comprises mitochondrialbiogenesis, peroxisome proliferation, and activation of the stressproteome. In an aspect, a combination comprises mitochondrialbiogenesis, peroxisome proliferation, transcription and/or translationof genes and proteins encoded by (a) genes comprising heat shock andunfolded protein, (b) genes for autophagic responses, (c) genes forantioxidant responses, and (d) genes for the c-jun-N-terminal kinasepathway. In an aspect, a combination comprises mitochondrial biogenesis,activation of the stress proteome, and transcription and/or translationof genes and proteins encoded by (a) genes comprising heat shock andunfolded protein, (b) genes for autophagic responses, (c) genes forantioxidant responses, and (d) genes for the c-jun-N-terminal kinasepathway. In an aspect, the combination comprises peroxisomeproliferation, activation of the stress proteome, and transcriptionand/or translation of genes and proteins encoded by (a) genes comprisingheat shock and unfolded protein, (b) genes for autophagic responses, (c)genes for antioxidant responses, and (d) genes for the c-jun-N-terminalkinase pathway.

In a disclosed method of treating autism, genes for heat shock proteinscan be genes for heat shock protein 40, 70, and/or 90 family members. Inan aspect, the genes for heat shock proteins comprise genes for heatshock protein 40 family members. In an aspect, the genes for heat shockproteins comprise genes for heat shock protein 70 family members. In anaspect, the genes for heat shock proteins comprise genes for heat shockprotein 90 family members. In an aspect, the genes for heat shockproteins comprise genes for heat shock protein 40 and 70 family members.In an aspect, the genes for heat shock proteins comprise genes for heatshock protein 40 and 90 family members. In an aspect, the genes for heatshock proteins comprise genes for heat shock protein 70 and 90 familymembers. In an aspect, the genes for heat shock proteins comprise genesfor heat shock protein 40, heat shock protein 70, and heat shock protein90 family members.

In a disclosed method of treating autism, unfolded protein genes maycomprise glucose regulated protein 78 (BIP), protein kinase RNA-likeendoplasmic reticulum kinase, (PERK), inositol requiring 1 (IRE1),and/or activating transcription factor 6. In an aspect, the unfoldedprotein gene is glucose regulated protein 78 (BIP). In an aspect, theunfolded protein gene is PERK. In an aspect, the unfolded protein geneis inositol requiring 1 (IRE1). In an aspect, the unfolded protein geneis transcription factor 6.

In an aspect of a disclosed method of treating autism, the unfoldedprotein genes comprise a combination of glucose regulated protein 78(BIP), protein kinase RNA-like endoplasmic reticulum kinase (PERK),inositol requiring 1 (IRE1), and/or activating transcription factor 6.In an aspect, the combination of unfolded protein genes comprisesglucose regulated protein 78 (BIP) and PERK. In an aspect, thecombination of unfolded protein genes comprises glucose regulatedprotein 78 (BIP) and inositol requiring 1 (IRE1). In an aspect, thecombination of unfolded protein genes comprises glucose regulatedprotein 78 (BIP) and activating transcription factor 6. In an aspect,the combination of unfolded protein genes comprises PERK and inositolrequiring 1 (IRE1). In an aspect, the combination of unfolded proteingenes comprises PERK and activating transcription factor 6. In anaspect, the combination of unfolded protein genes comprise glucoseregulated protein 78 (BIP), PERK, and inositol requiring 1 (IRE1). In anaspect of a disclosed method of treating autism, the unfolded proteingenes comprise a combination of glucose regulated protein 78 (BIP),PERK, and activating transcription factor 6. In an aspect of a disclosedmethod of treating autism, the unfolded protein genes comprise acombination of glucose regulated protein 78 (BIP), inositol requiring 1(IRE1), and activating transcription factor 6. In an aspect of adisclosed method of treating autism, the unfolded protein genes comprisea combination of PERK, inositol requiring 1 (IRE1), and activatingtranscription factor 6.

In a disclosed method of treating autism, autophagic response genes maycomprise beclin-1 (BCN1), autophagy protein 5 (ATG5), and/ormicrotubule-associated protein 1 light chain 3 (LC3 or APG8). In anaspect, the autophagic response gene is beclin-1 (BCN1). In an aspect,the autophagic response gene is autophagy protein 5 (ATG5). In anaspect, the autophagic response gene is microtubule-associated protein 1light chain 3 (LC3 or APG8). In an aspect, the autophagic response genescomprise a combination of beclin-1 (BCN1), autophagy protein 5 (ATG5),and/or microtubule-associated protein 1 light chain 3 (LC3 or APG8). Inan aspect, the combination comprises beclin-1 (BCN1 and autophagyprotein 5 (ATG5). In an aspect, the combination comprises beclin-1(BCN1) and microtubule-associated protein 1 light chain 3 (LC3 or APG8).In an aspect, the combination comprises autophagy protein 5 (ATG5) andmicrotubule-associated protein 1 light chain 3 (LC3 or APG8).

In a disclosed method of treating autism, antioxidant response genes maycomprise expression of nuclear factor erythroid 2-like 2 (NFE2L2), hemeoxygenase 1 (HMOX1), and superoxide dismutase 2 (SOD2). In an aspect,the antioxidant response gene is nuclear factor erythroid 2-like 2(NFE2L2). In an aspect, the antioxidant response gene is heme oxygenase1 (HMOX1). In an aspect, the antioxidant response gene is superoxidedismutase 2 (SOD2). In an aspect, the antioxidant response genescomprise expression of a combination of nuclear factor erythroid 2-like2 (NFE2L2), heme oxygenase 1 (HMOX1), and/or superoxide dismutase 2(SOD2). In an aspect, the antioxidant response genes comprise expressionof a combination of nuclear factor erythroid 2-like 2 (NFE2L2) and hemeoxygenase 1 (HMOX1). In an aspect, the antioxidant response genescomprise expression of a combination of nuclear factor erythroid 2-like2 (NFE2L2 and superoxide dismutase 2 (SOD2). In an aspect, theantioxidant response genes comprise expression of a combination of hemeoxygenase 1 (HMOX1) and superoxide dismutase 2 (SOD2). In an aspect, theantioxidant response genes comprise expression of a combination ofnuclear factor erythroid 2-like 2 (NFE2L2), heme oxygenase 1 (HMOX1),and superoxide dismutase 2 (SOD2).

In a disclosed method of treating autism, at least one cell of thesubject is located in the brain of the subject. In an aspect, at leastone cell of the subject is not in the brain of the subject. In anaspect, a general stress response occurs in all cells of the subject,but to differing degrees. In an aspect, the degree to which a celldemonstrates a general stress response depends on the specific tissueand cell type. For example, in an aspect, cells in the subject's brainare sensitive due to a high energy requirement and mitochondrialactivity.

In a disclosed method of treating autism, a composition comprises one ormore histone deacetylase inhibitors. In a disclosed method of treatingautism, a composition comprises one or more Class I histone deacetylaseinhibitors. For example, in an aspect, the composition comprises4-phenylbutyrate. In an aspect, the composition comprises sodiumbutyrate. In an aspect, the composition comprises trichostatin A. In anaspect, the composition comprises a combination of 4-phenylbutyrate,sodium butyrate, and/or trichostatin A. For example, the compositioncomprises a combination comprising phenylbutyrate and sodium butyrate.In an aspect, the composition comprises a combination comprisingphenylbutyrate and trichostatin A. In an aspect, the compositioncomprises a combination comprising sodium butyrate and trichostatin A.In an aspect, the composition comprises a combination comprisingphenylbutyrate, sodium butyrate, and trichostatin A.

In a disclosed method of treating autism, a composition compriseshydroxyurea.

In a disclosed method of treating autism, a composition comprises one ormore Class II histone deacetylase inhibitors.

In a disclosed method of treating autism, a composition comprisessulforaphane or a sulforaphane derivative or analog, or combinationsthereof.

In a disclosed method of treating autism, the composition comprises asulforaphane dithocarbamate metabolite.

In a disclosed method of treating autism, a composition comprises atleast one of a histone deacetylase inhibitor, a Class I histonedeacetylase inhibitor, a Class II histone deacetylase inhibitor,4-phenylbutyrate, trichostatin A, hydroxyurea, sulforaphane or asulforaphane analog, or a sulforaphane dithocarbamate metabolite. In anaspect, a composition comprises a combination of a histone deacetylaseinhibitor, a Class I histone deacetylase inhibitor, a Class II histonedeacetylase inhibitor, 4-phenylbutyrate, trichostatin A, hydroxyurea,sulforaphane or a sulforaphane analog, or a sulforaphane dithocarbamatemetabolite. In an aspect, the composition comprises a combination of ahistone deacetylase inhibitor, such as for example, a Class I histonedeacetylase inhibitor or a Class II histone deacetylase inhibitor, andone or more of the following: 4-phenylbutyrate, trichostatin A,hydroxyurea, sulforaphane or a sulforaphane analog, or a sulforaphanedithocarbamate metabolite. In an aspect, the composition comprises acombination of 4-phenylbutyrate, trichostatin A, hydroxyurea, andsulforaphane or a sulforaphane analog.

In a disclosed method of treating autism, the composition comprises acompound that is present as a structure represented by a formula:

or a subgroup or pharmaceutically acceptable salt thereof.

In a disclosed method of treating autism, the composition furthercomprises pharmaceutical excipients or diluents, or components offormulations used in delivery vehicles for appropriate administration ofthe composition.

In a disclosed method of treating autism, the composition isadministered orally, topically, by injection, intravascular,subcutaneously, intramuscularly, nasally, or by other known routes ofadministration. In a disclosed method of treating autism, thecomposition is administered one or more times. For example, in anaspect, the composition is administered at least one time per day. In anaspect, the composition is administered continuously. In an aspect, thecomposition is administered intermittently. In an aspect, administrationcan be repeated, for example, once per day, or two or more times perday, or once per week, or two or more times per week, or every otherweek, or once per month, or one or more times per month, or every otherday, or every other week, or every over month, or every other year, soforth and so on.

A disclosed method of treating autism can further comprise evaluatingthe progression of a subject's neurological disease or neurologicaldisorder, such as, for example, autism or one or more autism spectrumdisorders. A clinician (e.g., a physician) or researcher can evaluatingthe subject at scheduled times. For example, in an aspect, theacquisition of data for a human subject can be performed periodically,wherein the scheduled times occur at regular intervals, such as every 3months, 6 months, 9 months, or every year, every other year, every 5years, every 10 years for the life of the subject. In an aspect, thescheduled times need not be periodic. For another example, in an aspect,the acquisition of data for a non-human subject can be carried outperiodically at scheduled times spaced at regular intervals, such asevery week, every other week, every month, every other month, every 3months, every 6 months, every 9 months, every year, every other year forthe life of the non-human subject.

C. Methods of Treating Autism Spectrum Disorder

Disclosed herein are methods of treating one or more autism spectrumdisorders.

Disclosed herein is a method of treating one or more autism spectrumdisorders comprising administering to a subject diagnosed with one ormore autism spectrum disorders an effective amount of a pharmaceuticalcomposition comprising a compound that induces a general cellular stressresponse in at least one cell of the person.

Disclosed herein is a method of treating one or more autism spectrumdisorders comprising administering to a subject diagnosed with one ormore autism spectrum disorders an effective amount of a pharmaceuticalcomposition that modulates measurable effects of behavioral symptoms.

Disclosed herein is a method of treating one or more autism spectrumdisorders comprising administering to a subject diagnosed with one ormore autism spectrum disorders an effective amount of a pharmaceuticalcomposition that modulates social responsiveness of the subject treated.The person skilled in the art is familiar with the social responsivenessof a subject with autism or autism spectrum disorder as discussed inConstantino et al., 2003 (J Autism Devel Disorders, 33: 427-433, whichdiscusses the Social Responsiveness Scale or SRS—a well-validatedmeasure of autistic traits).

In a disclosed method of treating one or more autism spectrum disorders,the subject is male or female. In an aspect, the subject does not denotea particular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are intended to be covered. In anaspect, the subject is a mammal.

In a disclosed method of treating one or more autism spectrum disorders,in the at least one cell of the subject, the stress proteome isstimulated. In a disclosed method of treating autism, in the at leastone cell of the subject, increased nitrous oxide production is measured.

In a disclosed method of treating one or more autism spectrum disorders,in the at least one cell of the subject, stress-sensing organelles areincreased from an amount prior to the administration of the composition.A stress-sensing organelle can be a mitrochondrion or a peroxisome. Inan aspect, a stress-sensing organelle is a mitochondrion. In an aspect,a stress-sensing organelle is a peroxisome.

In a disclosed method of treating one or more autism spectrum disorders,a general cellular stress response of a disclosed method of treatingautism comprises at least one of the following: (1) mitochondrialbiogenesis, (2) peroxisome proliferation, (3) activation of the stressproteome, or (4) transcription and/or translation of genes and proteinsencoded by (a) genes comprising heat shock and unfolded protein, (b)genes for autophagic responses, (c) genes for antioxidant responses, and(d) genes for the c-jun-N-terminal kinase pathway. In an aspect, ageneral cellular stress response comprises more than one of thefollowing: (1) mitochondrial biogenesis, (2) peroxisome proliferation,(3) activation of the stress proteome, or (4) transcription and/ortranslation of genes and proteins encoded by (a) genes comprising heatshock and unfolded protein, (b) genes for autophagic responses, (c)genes for antioxidant responses, and (d) genes for the c-jun-N-terminalkinase pathway.

For example, in an aspect, a combination comprises mitochondrialbiogenesis and peroxisome proliferation. In an aspect, the combinationcomprises mitochondrial biogenesis and activation of the stressproteome. In an aspect, the combination comprises mitochondrialbiogenesis and transcription and/or translation of genes and proteinsencoded by (a) genes comprising heat shock and unfolded protein, (b)genes for autophagic responses, (c) genes for antioxidant responses, and(d) genes for the c-jun-N-terminal kinase pathway. In an aspect, thecombination comprises peroxisome proliferation and activation of thestress proteome.

In an aspect, the combination comprises peroxisome proliferation andtranscription and/or translation of genes and proteins encoded by (a)genes comprising heat shock and unfolded protein, (b) genes forautophagic responses, (c) genes for antioxidant responses, and (d) genesfor the c-jun-N-terminal kinase pathway. In an aspect, the combinationcomprises activation of the stress proteome and transcription and/ortranslation of genes and proteins encoded by (a) genes comprising heatshock and unfolded protein, (b) genes for autophagic responses, (c)genes for antioxidant responses, and (d) genes for the c-jun-N-terminalkinase pathway. In an aspect, the combination comprises mitochondrialbiogenesis, peroxisome proliferation, and activation of the stressproteome. In an aspect, the combination comprises mitochondrialbiogenesis, peroxisome proliferation, transcription and/or translationof genes and proteins encoded by (a) genes comprising heat shock andunfolded protein, (b) genes for autophagic responses, (c) genes forantioxidant responses, and (d) genes for the c-jun-N-terminal kinasepathway. In an aspect, the combination comprises mitochondrialbiogenesis, activation of the stress proteome, and transcription and/ortranslation of genes and proteins encoded by (a) genes comprising heatshock and unfolded protein, (b) genes for autophagic responses, (c)genes for antioxidant responses, and (d) genes for the c-jun-N-terminalkinase pathway. In an aspect, the combination comprises peroxisomeproliferation, activation of the stress proteome, and transcriptionand/or translation of genes and proteins encoded by (a) genes comprisingheat shock and unfolded protein, (b) genes for autophagic responses, (c)genes for antioxidant responses, and (d) genes for the c-jun-N-terminalkinase pathway.

In a disclosed method of treating one or more autism spectrum disorders,genes for heat shock proteins can be genes for heat shock protein 40,70, and/or 90 family members. In an aspect, the genes for heat shockproteins comprise genes for heat shock protein 40 family members. In anaspect, the genes for heat shock proteins comprise genes for heat shockprotein 70 family members. In an aspect, the genes for heat shockproteins comprise genes for heat shock protein 90 family members. In anaspect, the genes for heat shock proteins comprise genes for heat shockprotein 40 and 70 family members. In an aspect, the genes for heat shockproteins comprise genes for heat shock protein 40 and 90 family members.In an aspect, the genes for heat shock proteins comprise genes for heatshock protein 70 and 90 family members. In an aspect, the genes for heatshock proteins comprise genes for heat shock protein 40, heat shockprotein 70, and heat shock protein 90 family members.

In a disclosed method of treating one or more autism spectrum disorders,unfolded protein genes comprise glucose regulated protein 78 (BIP),protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositolrequiring 1 (IRE1), and/or activating transcription factor 6. In anaspect, the unfolded protein gene is glucose regulated protein 78 (BIP).In an aspect, the unfolded protein gene is PERK. In an aspect, theunfolded protein gene is inositol requiring 1 (IRE1). In an aspect, theunfolded protein gene is transcription factor 6.

In an aspect of a disclosed method of treating one or more autismspectrum disorders, the unfolded protein genes comprise a combination ofglucose regulated protein 78 (BIP), protein kinase RNA-like endoplasmicreticulum kinase (PERK), inositol requiring 1 (IRE1), and/or activatingtranscription factor 6. In an aspect, the combination of unfoldedprotein genes comprises glucose regulated protein 78 (BIP) and PERK. Inan aspect, the combination of unfolded protein genes comprises glucoseregulated protein 78 (BIP) and inositol requiring 1 (IRE1). In anaspect, the combination of unfolded protein genes comprises glucoseregulated protein 78 (BIP) and activating transcription factor 6. In anaspect, the combination of unfolded protein genes comprises PERK andinositol requiring 1 (IRE1). In an aspect, the combination of unfoldedprotein genes comprises PERK and activating transcription factor 6. Inan aspect, the combination of unfolded protein genes comprise glucoseregulated protein 78 (BIP), PERK, and inositol requiring 1 (IRE1). In anaspect of a disclosed method of treating autism, the unfolded proteingenes comprise a combination of glucose regulated protein 78 (BIP),PERK, and activating transcription factor 6. In an aspect of a disclosedmethod of treating autism, the unfolded protein genes comprise acombination of glucose regulated protein 78 (BIP), inositol requiring 1(IRE1), and activating transcription factor 6. In an aspect of adisclosed method of treating autism, the unfolded protein genes comprisea combination of PERK, inositol requiring 1 (IRE1), and activatingtranscription factor 6.

In a disclosed method of treating one or more autism spectrum disorders,the autophagic response genes comprise beclin-1 (BCN1), autophagyprotein 5 (ATG5), and/or microtubule-associated protein 1 light chain 3(LC3 or APG8). In an aspect, the autophagic response gene is beclin-1(BCN1). In an aspect, the autophagic response gene is autophagy protein5 (ATG5). In an aspect, the autophagic response gene ismicrotubule-associated protein 1 light chain 3 (LC3 or APG8). In anaspect, the autophagic response genes comprise a combination of beclin-1(BCN1), autophagy protein 5 (ATG5), and/or microtubule-associatedprotein 1 light chain 3 (LC3 or APG8). In an aspect, the combinationcomprises beclin-1 (BCN1 and autophagy protein 5 (ATG5). In an aspect,the combination comprises beclin-1 (BCN1) and microtubule-associatedprotein 1 light chain 3 (LC3 or APG8). In an aspect, the combinationcomprises autophagy protein 5 (ATG5) and microtubule-associated protein1 light chain 3 (LC3 or APG8).

In a disclosed method of treating one or more autism spectrum disorders,the antioxidant response genes comprise expression of nuclear factorerythroid 2-like 2 (NFE2L2), heme oxygenase 1 (HMOX1), and superoxidedismutase 2 (SOD2). In an aspect, the antioxidant response gene isnuclear factor erythroid 2-like 2 (NFE2L2). In an aspect, theantioxidant response gene is heme oxygenase 1 (HMOX1). In an aspect, theantioxidant response gene is superoxide dismutase 2 (SOD2). In anaspect, the antioxidant response genes comprise expression of acombination of nuclear factor erythroid 2-like 2 (NFE2L2), hemeoxygenase 1 (HMOX1), and/or superoxide dismutase 2 (SOD2). In an aspect,the antioxidant response genes comprise expression of a combination ofnuclear factor erythroid 2-like 2 (NFE2L2) and heme oxygenase 1 (HMOX1).In an aspect, the antioxidant response genes comprise expression of acombination of nuclear factor erythroid 2-like 2 (NFE2L2 and superoxidedismutase 2 (SOD2). In an aspect, the antioxidant response genescomprise expression of a combination of heme oxygenase 1 (HMOX1) andsuperoxide dismutase 2 (SOD2). In an aspect, the antioxidant responsegenes comprise expression of a combination of nuclear factor erythroid2-like 2 (NFE2L2), heme oxygenase 1 (HMOX1), and superoxide dismutase 2(SOD2).

In a disclosed method of treating one or more autism spectrum disorders,the at least one cell of the subject is located in the brain of thesubject. In an aspect, the at least one cell of the subject is not inthe brain of the subject. In an aspect, a general stress response occursin all cells of the subject, but to differing degrees. In an aspect, thedegree to which a cell demonstrates a general stress response depends onthe specific tissue and cell type. For example, in an aspect, cells inthe subject's brain are especially sensitive due to a high energyrequirement and mitochondrial activity.

In a disclosed method of treating one or more autism spectrum disorders,the composition comprises one or more Class I histone deacetylaseinhibitors. For example, in an aspect, the composition comprises4-phenylbutyrate. In an aspect, the composition comprises sodiumbutyrate. In an aspect, the composition comprises trichostatin A. In anaspect, the composition comprises a combination of 4-phenylbutyrate,sodium butyrate, and/or trichostatin A. For example, the compositioncomprises a combination comprising phenylbutyrate and sodium butyrate.In an aspect, the composition comprises a combination comprisingphenylbutyrate and trichostatin A. In an aspect, the compositioncomprises a combination comprising sodium butyrate and trichostatin A.In an aspect, the composition comprises a combination comprisingphenylbutyrate, sodium butyrate, and trichostatin A.

In a disclosed method of treating one or more autism spectrum disorders,the composition comprises hydroxyurea.

In a disclosed method of treating one or more autism spectrum disorders,the composition comprises one or more Class II histone deacetylaseinhibitors.

In a disclosed method of treating one or more autism spectrum disorders,the composition comprises sulforaphane or a sulforaphane derivative oranalog.

In a disclosed method of treating one or more autism spectrum disorders,the composition comprises a sulforaphane dithocarbamate metabolite.

In a disclosed method of treating one or more autism spectrum disorders,the composition comprises at least one of a histone deacetylaseinhibitor, a Class I histone deacetylase inhibitor, a Class II histonedeacetylase inhibitor, 4-phenylbutyrate, trichostatin A, hydroxyurea,sulforaphane or a sulforaphane analog, or a sulforaphane dithocarbamatemetabolite. In an aspect, the composition comprises a combination of ahistone deacetylase inhibitor, a Class I histone deacetylase inhibitor,a Class II histone deacetylase inhibitor, 4-phenylbutyrate, trichostatinA, hydroxyurea, sulforaphane or a sulforaphane analog, or a sulforaphanedithocarbamate metabolite. In an aspect, the composition comprises acombination of a histone deacetylase inhibitor, such as for example, aClass I histone deacetylase inhibitor or a Class II histone deacetylaseinhibitor, and one or more of the following: 4-phenylbutyrate,trichostatin A, hydroxyurea, sulforaphane or a sulforaphane analog, or asulforaphane dithocarbamate metabolite. In an aspect, the compositioncomprises a combination of 4-phenylbutyrate, trichostatin A,hydroxyurea, and sulforaphane or a sulforaphane analog.

In a disclosed method of treating one or more autism spectrum disorders,the composition comprises a compound that is present as a structurerepresented by a formula:

or a subgroup or pharmaceutically acceptable salt thereof.

In a disclosed method of treating one or more autism spectrum disorders,the composition further comprises pharmaceutical excipients or diluents,or components of formulations used in delivery vehicles for appropriateadministration of the composition.

In a disclosed method of treating one or more autism spectrum disorders,the composition is administered orally, topically, by injection,nasally, or by other known routes of administration.

In a disclosed method of treating one or more autism spectrum disorders,the composition is administered one or more times. For example, in anaspect, the composition is administered at least one time per day. In anaspect, the composition is administered continuously. In an aspect, thecomposition is administered intermittently. In an aspect, administrationcan be repeated, for example, once per day, or two or more times perday, or once per week, or two or more times per week, or every otherweek, or once per month, or one or more times per month, or every otherday, or every other week, or every over month, or every other year, soforth and so on.

A disclosed method of treating one or more autism spectrum disorders canfurther comprise evaluating the progression of a subject's neurologicaldisease or neurological disorder, such as, for example, autism or one ormore autism spectrum disorders. A clinician (e.g., a physician) orresearcher can evaluating the subject at scheduled times. For example,in an aspect, the acquisition of data for a human subject can beperformed periodically, wherein the scheduled times occur at regularintervals, such as every 3 months, 6 months, 9 months, or every year,every other year, every 5 years, every 10 years for the life of thesubject. In an aspect, the scheduled times need not be periodic. Foranother example, in an aspect, the acquisition of data for a non-humansubject can be carried out periodically at scheduled times spaced atregular intervals, such as every week, every other week, every month,every other month, every 3 months, every 6 months, every 9 months, everyyear, every other year for the life of the non-human subject.

D. Methods of Determining Effectiveness of a Compound

Disclosed herein are methods of determining the effectiveness of acompound in treating autism or an autism related disorder comprisingadministering to a first cell an effective amount of a compound to betested, comparing the response of the first cell with the response of anidentical cell that was treated with a compound that induces ageneralized stress response, and determining whether or not the testedcompound induces a general cellular stress response in cells. In anaspect, the cell is a normal human fibroblast. In an aspect, the cell isXALD fibroblast. In an aspect, the cell is a K562 cell.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the cell is from asubject. In an aspect, the subject is male or female. In an aspect, thesubject does not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. In an aspect, the subject is a mammal.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the stress proteome ofthe first cell is stimulated. In a disclosed method of determining theeffectiveness of a compound in treating autism or an autism relateddisorder, increased nitrous oxide production is measured in the firstcell.

In a disclosed method of In a disclosed method of determining theeffectiveness of a compound in treating autism or an autism relateddisorder, stress-sensing organelles are increased in the first cell froman amount prior to the administration of the compound. A stress-sensingorganelle can be a mitrochondrion or a peroxisome. In an aspect, astress-sensing organelle is a mitochondrion. In an aspect, astress-sensing organelle is a peroxisome.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, a general cellular stressresponse comprises at least one of the following: (1) mitochondrialbiogenesis, (2) peroxisome proliferation, (3) activation of the stressproteome, or (4) transcription and/or translation of genes and proteinsencoded by (a) genes comprising heat shock and unfolded protein, (b)genes for autophagic responses, (c) genes for antioxidant responses, and(d) genes for the c-jun-N-terminal kinase pathway. In an aspect, ageneral cellular stress response comprises more than one of thefollowing: (1) mitochondrial biogenesis, (2) peroxisome proliferation,(3) activation of the stress proteome, or (4) transcription and/ortranslation of genes and proteins encoded by (a) genes comprising heatshock and unfolded protein, (b) genes for autophagic responses, (c)genes for antioxidant responses, and (d) genes for the c-jun-N-terminalkinase pathway.

For example, in an aspect, the combination comprises mitochondrialbiogenesis and peroxisome proliferation. In an aspect, the combinationcomprises mitochondrial biogenesis and activation of the stressproteome. In an aspect, the combination comprises mitochondrialbiogenesis and transcription and/or translation of genes and proteinsencoded by (a) genes comprising heat shock and unfolded protein, (b)genes for autophagic responses, (c) genes for antioxidant responses, and(d) genes for the c-jun-N-terminal kinase pathway. In an aspect, thecombination comprises peroxisome proliferation and activation of thestress proteome.

In an aspect, the combination comprises peroxisome proliferation andtranscription and/or translation of genes and proteins encoded by (a)genes comprising heat shock and unfolded protein, (b) genes forautophagic responses, (c) genes for antioxidant responses, and (d) genesfor the c-jun-N-terminal kinase pathway. In an aspect, the combinationcomprises activation of the stress proteome and transcription and/ortranslation of genes and proteins encoded by (a) genes comprising heatshock and unfolded protein, (b) genes for autophagic responses, (c)genes for antioxidant responses, and (d) genes for the c-jun-N-terminalkinase pathway. In an aspect, the combination comprises mitochondrialbiogenesis, peroxisome proliferation, and activation of the stressproteome. In an aspect, the combination comprises mitochondrialbiogenesis, peroxisome proliferation, transcription and/or translationof genes and proteins encoded by (a) genes comprising heat shock andunfolded protein, (b) genes for autophagic responses, (c) genes forantioxidant responses, and (d) genes for the c-jun-N-terminal kinasepathway. In an aspect, the combination comprises mitochondrialbiogenesis, activation of the stress proteome, and transcription and/ortranslation of genes and proteins encoded by (a) genes comprising heatshock and unfolded protein, (b) genes for autophagic responses, (c)genes for antioxidant responses, and (d) genes for the c-jun-N-terminalkinase pathway. In an aspect, the combination comprises peroxisomeproliferation, activation of the stress proteome, and transcriptionand/or translation of genes and proteins encoded by (a) genes comprisingheat shock and unfolded protein, (b) genes for autophagic responses, (c)genes for antioxidant responses, and (d) genes for the c-jun-N-terminalkinase pathway.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the genes for heat shockproteins can be genes for heat shock protein 40, 70, and/or 90 familymembers. In an aspect, the genes for heat shock proteins comprise genesfor heat shock protein 40 family members. In an aspect, the genes forheat shock proteins comprise genes for heat shock protein 70 familymembers. In an aspect, the genes for heat shock proteins comprise genesfor heat shock protein 90 family members. In an aspect, the genes forheat shock proteins comprise genes for heat shock protein 40 and 70family members. In an aspect, the genes for heat shock proteins comprisegenes for heat shock protein 40 and 90 family members. In an aspect, thegenes for heat shock proteins comprise genes for heat shock protein 70and 90 family members. In an aspect, the genes for heat shock proteinscomprise genes for heat shock protein 40, heat shock protein 70, andheat shock protein 90 family members.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the unfolded proteingenes comprise glucose regulated protein 78 (BIP), protein kinaseRNA-like endoplasmic reticulum kinase (PERK), inositol requiring 1(IRE1), and/or activating transcription factor 6. In an aspect, theunfolded protein gene is glucose regulated protein 78 (BIP). In anaspect, the unfolded protein gene is PERK. In an aspect, the unfoldedprotein gene is inositol requiring 1 (IRE1). In an aspect, the unfoldedprotein gene is transcription factor 6.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the unfolded proteingenes comprise a combination of glucose regulated protein 78 (BIP),protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositolrequiring 1 (IRE1), and/or activating transcription factor 6. In anaspect, the combination of unfolded protein genes comprises glucoseregulated protein 78 (BIP) and PERK. In an aspect, the combination ofunfolded protein genes comprises glucose regulated protein 78 (BIP) andinositol requiring 1 (IRE1). In an aspect, the combination of unfoldedprotein genes comprises glucose regulated protein 78 (BIP) andactivating transcription factor 6. In an aspect, the combination ofunfolded protein genes comprises PERK and inositol requiring 1 (IRE1).In an aspect, the combination of unfolded protein genes comprises PERKand activating transcription factor 6. In an aspect, the combination ofunfolded protein genes comprise glucose regulated protein 78 (BIP),PERK, and inositol requiring 1 (IRE1). In an aspect of a disclosedmethod of treating autism, the unfolded protein genes comprise acombination of glucose regulated protein 78 (BIP), PERK, and activatingtranscription factor 6. In an aspect of a disclosed method of treatingautism, the unfolded protein genes comprise a combination of glucoseregulated protein 78 (BIP), inositol requiring 1 (IRE1), and activatingtranscription factor 6. In an aspect of a disclosed method of treatingautism, the unfolded protein genes comprise a combination of PERK,inositol requiring 1 (IRE1), and activating transcription factor 6.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the autophagic responsegenes comprise beclin-1 (BCN1), autophagy protein 5 (ATG5), and/ormicrotubule-associated protein 1 light chain 3 (LC3 or APG8). In anaspect, the autophagic response gene is beclin-1 (BCN1). In an aspect,the autophagic response gene is autophagy protein 5 (ATG5). In anaspect, the autophagic response gene is microtubule-associated protein 1light chain 3 (LC3 or APG8). In an aspect, the autophagic response genescomprise a combination of beclin-1 (BCN1), autophagy protein 5 (ATG5),and/or microtubule-associated protein 1 light chain 3 (LC3 or APG8). Inan aspect, the combination comprises beclin-1 (BCN1 and autophagyprotein 5 (ATG5). In an aspect, the combination comprises beclin-1(BCN1) and microtubule-associated protein 1 light chain 3 (LC3 or APG8).In an aspect, the combination comprises autophagy protein 5 (ATG5) andmicrotubule-associated protein 1 light chain 3 (LC3 or APG8).

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the antioxidant responsegenes comprise expression of nuclear factor erythroid 2-like 2 (NFE2L2),heme oxygenase 1 (HMOX1), and superoxide dismutase 2 (SOD2). In anaspect, the antioxidant response gene is nuclear factor erythroid 2-like2 (NFE2L2). In an aspect, the antioxidant response gene is hemeoxygenase 1 (HMOX1). In an aspect, the antioxidant response gene issuperoxide dismutase 2 (SOD2). In an aspect, the antioxidant responsegenes comprise expression of a combination of nuclear factor erythroid2-like 2 (NFE2L2), heme oxygenase 1 (HMOX1), and/or superoxide dismutase2 (SOD2). In an aspect, the antioxidant response genes compriseexpression of a combination of nuclear factor erythroid 2-like 2(NFE2L2) and heme oxygenase 1 (HMOX1). In an aspect, the antioxidantresponse genes comprise expression of a combination of nuclear factorerythroid 2-like 2 (NFE2L2 and superoxide dismutase 2 (SOD2). In anaspect, the antioxidant response genes comprise expression of acombination of heme oxygenase 1 (HMOX1) and superoxide dismutase 2(SOD2). In an aspect, the antioxidant response genes comprise expressionof a combination of nuclear factor erythroid 2-like 2 (NFE2L2), hemeoxygenase 1 (HMOX1), and superoxide dismutase 2 (SOD2).

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the first cell is a braincell. In an aspect, the first cell is not a brain cell. In an aspect, ageneral stress response occurs in all cells of a subject, but todiffering degrees. In an aspect, the degree to which a cell demonstratesa general stress response depends on the specific tissue and cell type.For example, in an aspect, a brain cell is especially sensitive due to ahigh energy requirement and mitochondrial activity.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the compound comprises aClass I histone deacetylase inhibitors. For example, in an aspect, thecompound comprises 4-phenylbutyrate. In an aspect, the compoundcomprises sodium butyrate. In an aspect, the compound comprisestrichostatin A. In an aspect, the compound comprises a combination of4-phenylbutyrate, sodium butyrate, and/or trichostatin A. For example,the compound comprises a combination comprising phenylbutyrate andsodium butyrate. In an aspect, the compound comprises a combinationcomprising phenylbutyrate and trichostatin A. In an aspect, the compoundcomprises a combination comprising sodium butyrate and trichostatin A.In an aspect, the compound comprises a combination comprisingphenylbutyrate, sodium butyrate, and trichostatin A.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the compound compriseshydroxyurea.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the compound comprisesone or more Class II histone deacetylase inhibitors.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the compound comprisessulforaphane or a sulforaphane analog.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the compound comprises asulforaphane dithocarbamate metabolite.

In a disclosed method of determining the effectiveness of a compound intreating autism or an autism related disorder, the compound comprises atleast one of a histone deacetylase inhibitor, a Class I histonedeacetylase inhibitor, a Class II histone deacetylase inhibitor,4-phenylbutyrate, trichostatin A, hydroxyurea, sulforaphane or asulforaphane analog, or a sulforaphane dithocarbamate metabolite. In anaspect, the composition comprises a combination of a histone deacetylaseinhibitor, a Class I histone deacetylase inhibitor, a Class II histonedeacetylase inhibitor, 4-phenylbutyrate, trichostatin A, hydroxyurea,sulforaphane or a sulforaphane analog, or a sulforaphane dithocarbamatemetabolite. In an aspect, the compound comprises a combination of ahistone deacetylase inhibitor, such as for example, a Class I histonedeacetylase inhibitor or a Class II histone deacetylase inhibitor, andone or more of the following: 4-phenylbutyrate, trichostatin A,hydroxyurea, sulforaphane or a sulforaphane analog, or a sulforaphanedithocarbamate metabolite. In an aspect, the compound comprises acombination of 4-phenylbutyrate, trichostatin A, hydroxyurea, andsulforaphane or a sulforaphane analog.

In a disclosed method of determining the effectiveness of a testcompound in treating autism or an autism related disorder, a comparativecompound, to which the compound being tested is compared to foreffectiveness or changes in the cells in the assay, is present as astructure represented by a formula:

or a subgroup or pharmaceutically acceptable salt thereof.

Compounds may be tested in in vitro conditions or in in vivo conditions.

E. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

As used herein, a cruciferous sprout is a plant or seedling that is atan early stage of development following seed germination. Cruciferousseeds are placed in an environment in which they germinate and grow. Thecruciferous sprouts may be harvested following seed germination throughand including the 2-leaf stage.

As used herein, the term “subject” refers to the target ofadministration, e.g., an animal. Thus, the subject of the hereindisclosed methods can be a vertebrate, such as a mammal, a fish, a bird,a reptile, or an amphibian. Alternatively, the subject of the hereindisclosed methods can be a human, non-human primate, horse, pig, rabbit,dog, sheep, goat, cow, cat, guinea pig or rodent. The term does notdenote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Inone aspect, the subject is a mammal A subject also can be a transgenic,non-human animal including but not limited to a transgenic mouse ortransgenic rat.

A patient refers to a subject afflicted with a disease or disorder. Theterm “patient” includes human and veterinary subjects. In some aspectsof the disclosed methods, the subject has been diagnosed with a need fortreatment for autism or one or more autism spectrum disorders prior tothe administering step.

As used herein, the term “analog” refers to a compound having astructure derived from the structure of a parent compound (e.g., acompound disclosed herein) and whose structure is sufficiently similarto those disclosed herein and based upon that similarity, would beexpected by one skilled in the art to exhibit the same or similaractivities and utilities as the claimed compounds, or to induce, as aprecursor, the same or similar activities and utilities as the claimedcompounds.

As used herein, “homolog” or “homologue” refers to a polypeptide ornucleic acid with homology to a specific known sequence. Specificallydisclosed are variants of the nucleic acids and polypeptides hereindisclosed which have at least 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percenthomology to the stated or known sequence. Those of skill in the artreadily understand how to determine the homology of two proteins ornucleic acids. For example, the homology can be calculated afteraligning the two sequences so that the homology is at its highest level.It is understood that one way to define any variants, modifications, orderivatives of the disclosed genes and proteins herein is throughdefining the variants, modification, and derivatives in terms ofhomology to specific known sequences.

As used herein, the term “treatment” refers to the medical management ofa subject or a patient with the intent to cure, ameliorate, stabilize,or prevent a disease, pathological condition, or disorder, such as, forexample, autism or one or more autism spectrum disorders. This termincludes active treatment, that is, treatment directed specificallytoward the improvement of a disease, pathological condition, ordisorder, and also includes causal treatment, that is, treatmentdirected toward removal of the cause of the associated disease,pathological condition, or disorder. In addition, this term includespalliative treatment, that is, treatment designed for the relief ofsymptoms rather than the curing of the disease, pathological condition,or disorder; preventative treatment, that is, treatment directed tominimizing or partially or completely inhibiting the development of theassociated disease, pathological condition, or disorder; and supportivetreatment, that is, treatment employed to supplement another specifictherapy directed toward the improvement of the associated disease,pathological condition, or disorder. In various aspects, the term coversany treatment of a subject, including a mammal (e.g., a human), andincludes: (i) preventing the disease from occurring in a subject thatcan be predisposed to the disease but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, i.e., arresting its development;or (iii) relieving the disease, i.e., causing regression of the disease.In an aspect, the disease, pathological condition, or disorder is autismor one or more autism spectrum disorders.

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve the desired result orto have an effect on an undesired condition. For example, a“therapeutically effective amount” refers to an amount that issufficient to achieve the desired therapeutic result or to have aneffect on undesired symptoms, but is generally insufficient to causeadverse side effects. The specific therapeutically effective dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration; the route ofadministration; the rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of a compound at levels lower than those required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved. If desired, the effective dailydose can be divided into multiple doses for purposes of administration.Consequently, single dose compositions can contain such amounts orsubmultiples thereof to make up the daily dose. The dosage can beadjusted by the individual physician in the event of anycontraindications. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days. Guidance canbe found in the literature for appropriate dosages for given classes ofpharmaceutical products. In further various aspects, a preparation canbe administered in a “prophylactically effective amount”; that is, anamount effective for prevention of a disease or condition.

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve the desired result orto have an effect on an undesired condition. For example, a“therapeutically effective amount” refers to an amount that issufficient to achieve the desired therapeutic result or to have aneffect on undesired symptoms, but is generally insufficient to causeadverse side effects. The specific therapeutically effective dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration; the route ofadministration; the rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed and like factors well known in themedical arts.

Unless otherwise expressly stated, it is in no way intended that anymethod or aspect set forth herein be construed as requiring that itssteps be performed in a specific order. Accordingly, where a methodclaim does not specifically state in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including matters of logic withrespect to arrangement of steps or operational flow, plain meaningderived from grammatical organization or punctuation, or the number ortype of aspects described in the specification.

As used herein, the amino acid abbreviations are conventional one lettercodes for the amino acids and are expressed as follows: A, alanine; B,asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate,glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine;K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q,glutamine; R, arginine; S, serine; T, threonine; V, valine; W,tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

“Peptide” as used herein refers to any peptide, oligopeptide,polypeptide, gene product, expression product, or protein. For example,a peptide can be an enzyme. A peptide is comprised of consecutive aminoacids. Polypeptides encompass naturally occurring or synthetic molecule,and may contain modified amino acids other than the 20 gene-encodedamino acids. Polypeptides can be modified by either natural processes,such as post-translational processing, or by chemical modificationtechniques which are well known in the art. Modifications can occuranywhere in the polypeptide, including the peptide backbone, the aminoacid side-chains and the amino or carboxyl termini. The same type ofmodification can be present in the same or varying degrees at severalsites in a given polypeptide.

In general, the biological activity or biological action of agene/nucleic acid or peptide refers to any function exhibited orperformed by the gene/nucleic acid or peptide that is ascribed to thenaturally occurring form of the gene/nucleic acid or peptide as measuredor observed in vivo (i.e., in the natural physiological environment ofthe gene/nucleic acid or peptide) or in vitro (i.e., under laboratoryconditions).

The term “enzyme” as used herein refers to any peptide that catalyzes achemical reaction of other substances without itself being destroyed oraltered upon completion of the reaction. Typically, a peptide havingenzymatic activity catalyzes the formation of one or more products fromone or more substrates. Such peptides can have any type of enzymaticactivity including, without limitation, the enzymatic activity orenzymatic activities associated with enzymes such as those disclosedherein.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e., without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner. As used herein, the term “pharmaceuticallyacceptable carrier” refers to sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, as well as sterile powders forreconstitution into sterile injectable solutions or dispersions justprior to use. Examples of suitable aqueous and nonaqueous carriers,diluents, solvents or vehicles include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol and the like),carboxymethylcellulose and suitable mixtures thereof, vegetable oils(such as olive oil) and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coatingmaterials such as lecithin, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants. Thesecompositions can also contain adjuvants such as preservatives, wettingagents, emulsifying agents and dispersing agents. Prevention of theaction of microorganisms can be ensured by the inclusion of variousantibacterial and antifungal agents such as paraben, chlorobutanol,phenol, sorbic acid and the like. It can also be desirable to includeisotonic agents such as sugars, sodium chloride and the like. Prolongedabsorption of the injectable pharmaceutical form can be brought about bythe inclusion of agents, such as aluminum monostearate and gelatin,which delay absorption. Injectable depot forms are made by formingmicroencapsule matrices of the drug in biodegradable polymers such aspolylactide-polyglycolide, poly(orthoesters) and poly(anhydrides).Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Depot injectable formulations are also prepared by entrapping the drugin liposomes or microemulsions which are compatible with body tissues.The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedia just prior to use. Suitable inert carriers can include sugars suchas lactose. Desirably, at least 95% by weight of the particles of theactive ingredient have an effective particle size in the range of 0.01to 10 micrometers.

As used herein, “EC₅₀,” is intended to refer to the concentration ordose of a substance (e.g., a compound or a drug) that is required for50% enhancement or activation of a biological process, or component of aprocess, including a protein, subunit, organelle, ribonucleoprotein,etc. EC₅₀ also refers to the concentration or dose of a substance thatis required for 50% enhancement or activation in vivo, as furtherdefined elsewhere herein. Alternatively, EC₅₀ can refer to theconcentration or dose of compound that provokes a response halfwaybetween the baseline and maximum response. The response can be measuredin an in vitro or in vivo system as is convenient and appropriate forthe biological response of interest. For example, the response can bemeasured in vitro using cultured brain cells or in an ex vivo organculture system with isolated brain cells. Alternatively, the responsecan be measured in vivo using an appropriate research model such asrodent, including mice and rats. As appropriate, the response can bemeasured in a transgenic or knockout mouse or rat wherein a gene orgenes has been introduced or knocked-out, as appropriate, to replicate adisease process.

As used herein, “IC₅₀,” is intended to refer to the concentration ordose of a substance (e.g., a compound or a drug) that is required for50% inhibition or diminution of a biological process, or component of aprocess, including a protein, subunit, organelle, ribonucleoprotein,etc. IC₅₀ also refers to the concentration or dose of a substance thatis required for 50% inhibition or diminution in vivo, as further definedelsewhere herein. Alternatively, IC₅₀ also refers to the half maximal(50%) inhibitory concentration (IC) or inhibitory dose of a substance.The response can be measured in an in vitro or in vivo system as isconvenient and appropriate for the biological response of interest. Forexample, the response can be measured in vitro using cultured braincells or in an ex vivo organ culture system with isolated brain cells.Alternatively, the response can be measured in vivo using an appropriateresearch model such as rodent, including mice and rats. As appropriate,the response can be measured in a transgenic or knockout mouse or ratwherein a gene or genes has been introduced or knocked-out, asappropriate, to replicate a disease process.

Cells can be obtained from commercial sources such as the American TypeCulture Collection (ATCC) and can be prokaryotic or eukaryotic. Cellscan be grown in liquid media culture or on tissue culture plates. Thegrowth conditions will be dependent upon the specific cells used andsuch conditions would be known to one of skill in the art. Transfectionand growth of host cells is described in Maniatis et al.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.“Exemplary” means “an example of” and is not intended to convey anindication of a preferred or ideal embodiment. “Such as” is not used ina restrictive sense, but for explanatory purposes.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the terms “transformation” and “transfection” mean theintroduction of a nucleic acid, e.g., an expression vector, into arecipient cell including introduction of a nucleic acid to thechromosomal DNA of said cell. The art is familiar with variouscompositions, methods, techniques, etc. used to effect the introductionof a nucleic acid into a recipient cell. The art is familiar with suchcompositions, methods, techniques, etc. for both eukaryotic andprokaryotic cells. The art is familiar with such compositions, methods,techniques, etc. for the optimization of the introduction and expressionof a nucleic acid into and within a recipient cell.

The term “contacting” as used herein refers to bringing a disclosedcompound and a cell, target receptor, gene, peptide, or other biologicalentity together in such a manner that the compound can affect theactivity of the target (e.g., receptor, transcription factor, cell,etc.), either directly; i.e., by interacting with the target itself, orindirectly; i.e., by interacting with another molecule, co-factor,factor, or protein on which the activity of the target is dependent.

As used herein, the term “determining” can refer to measuring orascertaining a quantity or an amount or a change in expression and/oractivity level, e.g., of a nucleotide or transcript or polypeptide. Forexample, determining the amount of a disclosed transcript or polypeptidein a sample as used herein can refer to the steps that the skilledperson would take to measure or ascertain some quantifiable value of thetranscript or polypeptide in the sample. The art is familiar with theways to measure an amount of the disclosed nucleotides, transcripts,polypeptides, etc.

As used herein, the term “level” refers to the amount of a targetmolecule in a sample, e.g., a sample from a subject. The amount of themolecule can be determined by any method known in the art and willdepend in part on the nature of the molecule (i.e., gene, mRNA, cDNA,protein, enzyme, etc.). The art is familiar with quantification methodsfor nucleotides (e.g., genes, cDNA, mRNA, etc.) as well as proteins,polypeptides, enzymes, etc. It is understood that the amount or level ofa molecule in a sample need not be determined in absolute terms, but canbe determined in relative terms (e.g., when compare to a control or asham or an untreated sample).

By “modulate” is meant to alter, by increase or decrease. As usedherein, a “modulator” can mean a composition that can either increase ordecrease the expression level or activity level of a gene or geneproduct such as a peptide. Modulation in expression or activity does nothave to be complete. For example, expression or activity can bemodulated by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, 100% or any percentage in between as compared to a control cellwherein the expression or activity of a gene or gene product has notbeen modulated by the composition.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated orameliorated or detected or lessened by the compounds, compositions, ormethods disclosed herein. For example, “diagnosed with autism” or“diagnosed with one or more autism spectrum disorders” means having beensubjected to a physical examination by a person of skill, for example, aphysician, and found to have a condition that can be diagnosed ortreated by a compound or composition that alleviates or amelioratesautism or one or more autism spectrum disorders.

As used herein, the phrase “identified to be in need of treatment for adisorder,” or the like, refers to selection of a subject based upon needfor treatment of the disorder. For example, a subject can be identifiedas having a need for treatment of a disorder (e.g., autism or an autismspectrum) based upon an earlier diagnosis by a person of skill andthereafter subjected to treatment for the disorder. It is contemplatedthat the identification can, in one aspect, be performed by a persondifferent from the person making the diagnosis. It is also contemplated,in a further aspect, that the administration can be performed by one whosubsequently performed the administration.

As used herein, the terms “administering” and “administration” refer toany method of providing a disclosed compositions or pharmaceuticalpreparation comprising a disclosed composition to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, intracardiac administration, intramyocardialadministration, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, sublingual administration, buccal administration, andparenteral administration, including injectable such as intravenousadministration, intra-arterial administration, intramuscularadministration, and subcutaneous administration. Administration can becontinuous or intermittent. Administration can be repeated, for example,once per day, or two or more times per day, or once per week, or two ormore times per week, or every other week, or once per month, or one ormore times per month, or every other day, or every other week, or everyover month, or every other year, so forth and so on. In various aspects,a preparation can be administered therapeutically; that is, administeredto treat an existing disease or condition. In further various aspects, apreparation can be administered prophylactically; that is, administeredfor prevention of a disease or condition.

Disclosed are the components to be used to prepare a composition of theinvention as well as the compositions themselves to be used within themethods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds cannot be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions of the invention. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods of theinvention.

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which canrequire independent confirmation.

F. Experimental

As each of the small molecules can produce similar outcomes in diverseMendelian and complex disorders, these small molecules likely inducecommon cellular effects. These molecules include two (2) histonedeacetylase inhibitors, 4-phenylbutyrate and trichostatin A, and two (2)small molecules without direct histone deacetylase inhibitor activity,hydroxyurea and sulforaphane. In some cases, the therapeutic effects ofhistone deacetylase inhibitors (HDACi) are attributed to an increase inexpression of genes related to the disease-causing gene. In theneurological disorder X-linked adrenoleukodystrophy (XALD), thepotentially beneficial reduction of very long chain fatty acid levels bytrichostatin A was not due to an increase in expression of acompensatory gene. Rather, the reduction in 4-phenylbutyrate wasaccompanied by an increase in proliferation of the key stress-sensingorganelles, i.e., the mitochondria and peroxisomes. These studiesexamine whether 4-phenylbutyrate, trichostatin A, hydroxyurea, andsulforaphane share a common cellular response, which includes byinduction of mitochondrial biogenesis, peroxisome proliferation,activation of the stress proteome, which are collectively referred to asadaptive cell survival response.

The studies described herein have identified the activation of theevolutionarily conserved stress proteome and mitochondrial biogenesis asthe common cellular responses to small molecule therapy. This series ofresponses may be a common basis of therapeutic action in variousdiseases. Modulation of this novel therapeutic target could broaden therange of treatable diseases and be used to optimize therapeutic smallmolecules or agents without directly targeting the causative geneticabnormalities.

i) Materials and Methods

a. Cell Culture

Primary human fibroblasts, HeLa cells (ATCC #CCL-2, a gift from Dr. HalDietz, Johns Hopkins University, Baltimore, Md.), and K562 cells (ATCC#CCL-243) were grown in minimal Eagle medium (MEM; Mediatech, Manassas,Va.), RPMI medium (Mediatech) supplemented with 10 mM HEPES and 1 mMsodium pyruvate, or RPMI, respectively, and supplemented with 10% fetalbovine serum, penicillin (100 U/mL), and streptomycin (100 U/ml).Fibroblasts (70-95% confluent) were treated with 5 mM 4PBA (1 mM for JNKinhibition studies), 600 μM HU, 200 nM TSA, or 5 μM SFN unless otherwisespecified. K562 cells were treated with 1.2 mM SB or 100 μM HU. Alldrugs were obtained from Sigma-Aldrich (St. Louis, Mo.). Theconcentration of each drug per cell type was titrated to allow 100%viability and to minimally affect growth rate. The duration of treatmentvaried by experiment as indicated in the figure legends.

b. HDAC Activity Assay

A colorimetric HDAC activity assay was performed in triplicate using100-200 μg of HeLa or fibroblast lysates following the manufacturer'sprotocol (K331; Biovision, Mountain View, Calif.).

c. Indirect Immunofluorescence

Performed as described in Watkins et al., 1995 using 4% formaldehyde, 1%Triton X-100, primary antibody anti-ATP5B (Millipore, Billerica, Mass.)or anti-ABCD3 (Invitrogen, Carlsbad, Calif.), and secondary antibodysheep anti-mouse IgG FITC-conjugated (Santa Cruz Biotechnology, SantaCruz, Calif.) or goat anti-rabbit IgG rhodamine-conjugated (JacksonImmunoResearch, West Grove, Pa.).

d. In-Cell Western Analysis

The immunostaining of primary human fibroblasts with anti-ATP5B, DRAQ5(Cell Signaling Technologies, Danvers, Mass.; a cellular DNA stain tonormalize cell number), and secondary antibody IRDye 800CW donkeyanti-mouse IgG (Li-cor Biosciences, Lincoln, Nebr.) was identical to theindirect immunofluorescence procedure described above and performed induplicate. Imaging and quantification were performed using the OdysseyImager (Li-cor Biosciences).

e. Inhibition Studies

Antimycin A (10 ng/mL), LY294002, PD98059, RO-31-8425, SB203580,SP600125, and L-NAME were obtained from Sigma. Compound C was from EMDBiosciences (Darmstadt, Germany). Human fibroblasts were treated witheach inhibitor. Then, 45-75 min later, either 4PBA, HU, TSA, or SFN wasadded. Cells were stained four to six days after treatment with one ofthe small molecules. As a control for inhibitors dissolved in DMSO,fibroblasts were treated with an equivalent amount of DMSO in theabsence of inhibitor.

f. Real-Time PCR Analysis (RT-PCR)

Following the manufacturer's protocols, DNase I-treated cDNA wassynthesized using Superscript III or Thermoscript reverse transcriptase(Invitrogen). PCR reactions were performed in duplicate on the RocheLightcycler 3.5 (Basel, Switzerland) or the Bio-Rad iCycler (Hercules,Calif.) using Quantitech SYBR green PCR mix (Qiagen, Valencia, Calif.).GeNorm software calculated a normalization factor for each sample usingthe relative amounts of at least two of the following reference genes,β2-microglobulin, glyceraldehyde-6-phosphate, actin, and/or eukaryoticelongation factor 1A (Vandesompele et al., 2002). Primers for thevarious experiments described herein are listed below.

SEQ ID Primer Primer Sequence NO: Name (5′ to 3′) 1 MAP3K4FCCCAGAAACTTGGACTGGAA 2 MAP3K4R CCCAGCGCTAAGAGTAAACG 3 cJun-ex1FCAGGTGGCACAGCTTAAACA 4 cJun-ex1R TGAGTTGGCACCCACTGTTA 5 PGC1αFAGCTGCTGAAGAGGCAAGAG 6 PGC1αR CTCCAGGAAAAGCAAAGCTG 7 PGC1β-ex12FTGAAGCCATGGATTTTGACA 8 PGC1β-ex12R TATTGGAAGGGCCTTGTCTG 9 NRF1-ex11FTATCAGACAGCGCAGTCACC 10 NRF1-ex11R CAATGTCACCACCTCCACAG 11 NRF2-ex15FGGCAGCTGGTTTTATTGGAA 12 NRF2-ex15R ACAGTTTCACGTCCCCACTC 13 TFAM-ex7GCACAGGAAACCAGTTAGG 14 TFAM-ex7R ATCTGGGTTTTCCAAAGCAA 15 ATF4-ex2FAGATGACCTGGAAACCATGC 16 ATF4-ex2R GTGTCATCCAACGTGGTCAG 17 XBP1-ex6FACTGCCTGGAGGATAGCAGA 18 XBP1-ex6R ACCTTGGACTGCTGGATGTC 19 BiP-ex8FAATGACCAGAATCGCCTGAC 20 BiP-ex8R CGCTCCTTGAGCTTTTTGTC 21 HSF1FCCGCTCCACAGAGATACACA 22 HSF1R GTCTTGTCCGTCCATCCACT 23 ATF6FGCAGAACCTCAGCCACTTTC 24 ATF6R ACCGAGGAGACGAGACTGAA 25 HSP90AA1FGGCAGAGGCTGATAAGAACG 26 HSP90AA1R AGACAGGAGCGCAGTTTCAT 27 HSPA1AFCCGAGAAGGACGAGTTTGAG 28 HSPA1AR CTGGTACAGTCCGCTGATGA 29 DNAJC3FGATATTTTGCCCAGCAGGAA 30 DNAJC3R TTTGTCTCCCGTTTTGGAAC 31 NFE2L2F-intronACACGGTCCACAGCTCATC 32 NFE2L2R-intron TCTTGCCTCCAAAGTATGTCAA 33HMOX1-ex5F TCCGATGGGTCCTTACACTC 34 HMOX1-ex5R TAAGGAAGCCAGCCAAGAGA 35SOD2-ex2F CCCTGGAACCTCACATCAAC 36 SOD2-ex3R CGTTAGGGCTGAGGTTTGTC 37B-actinF ACGTTGCTATCCAGGCTGTGCTAT 38 B-actinR CGGTGAGGATCTTCATGAGGTAGT39 EEF1AF TTGCCGCCAGAACACAG 40 EEF1AR ACTTGCCCGAATCTACGTGT 41 B2MFTGCTGTCTCCATGTTTGATGTATCT 42 B2MR TCTCTGCTCCCCACCTCTAAGT 43 CHOP*FGGAGCTGGAAGCCTGGTATG 44 CHOP*R GCTCTGGGAGGTGCTTGT 45 Beclin1F (BCN1)CAAGATCCTGGACCGTGTCA 46 Beclin1R (BCN1) TGGCCATTTCTGTGGACATCA 47AP5LF-intron TGCAGAAGAAAATGGATTTCG 48 APG5LR-intron ACTGTCCATCTGCAGCCAC49 FL-SMNF GCGATGATTCTGACATTTGG 50 FL-SMNR AATGAAGCCACAGCTTTATCA 51Total SMNF ATAATTCCCCCACCACCTC 52 Total SMNR CACCTTCCTTCTTTTTGATTTTGTC

g. Western Blot Analysis

Cell lysates were collected in mammalian protein extraction reagent(m-PER; Thermo Scientific, Rockford, Ill.) plus 1× protease inhibitor(Sigma-Aldrich) and 1× Halt phosphatase inhibitor (Thermo Scientific).Denatured SDS-PAGE and immunoblot analyses were performed using thefollowing antibodies: phosphorylated JNK and β-actin from Santa CruzBiotechnology, HSP70, HSP90, BIP, phosphorylated eIF2α, and XBP1 fromCell Signaling, APG8 (Abgent, San Diego, Calif.), and SOD2 (Stressgen,Ann Arbor, Mich.). Quantitation was performed using a Fuji IntelligentDark box II, FUJI LAS-1000 Lite software and Image Gauge v4.22 software(Tokyo, Japan).

h. Fatty Acid B-Oxidation

Fatty acid β-oxidation activity in XALD fibroblasts was determined bymeasuring their capacity to degrade 1-¹⁴C-labeled fatty acids towater-soluble products as described in McGuiness et al., 2003.

i. Flow Cytometry

K562 cells were incubated with 50 nM Mitotracker Green FM, 300 nMMitotracker Deep Red 633 (Invitrogen), or PBS as a negative control for15 min at 37° C. or stained with anti-Pex14 using Caltag Fix and Permreagents (Invitrogen) following the manufacturer's instructions. Stainedcells were suspended in 0.5 mL of 1% paraformaldehyde/PBS and subjectedto flow cytometric analysis (FACScan machine, Becton Dickinson, FranklinLakes, N.J.).

j. Measurement of Hemoglobin

K562 cells were suspended in 1.1 mL media and 240 μL of DAF [workingsolution 50 μL 2,7 diaminofluorene (DAF) stock (50 ng DAF in 5 mL 90%glacial acetic acid), 50 μL 30% hydrogen peroxide, and 2.5 ml 200 mMTris-HCl, pH 7.0]. DAF stained hemoglobinized cells were scored using ahemocytometer.

k. Mitotracker Staining

SMA type I (GM00232) fibroblasts were treated with 2.5 mM 4PBA, 300 mMHU, 100 nM TSA or 2.5 mM SFN for 5 days, live stained with 25 nMMitotracker Red CMXROS (Invitrogen) in pre-warmed MEM media for 15 minat 37° C. in a CO₂ incubator. Stained cells were washed with pre-warmedMEM, washed with PBS and excess PBS was removed. The cells were examinedunder the microscope at ×80.

l. SIRT1 Activity Assay

A fluorescence-based SIRT1 activity assay was performed in triplicatefollowing the manufacturer's protocol (10010401; Cayman ChemicalCompany, Ann Arbor, Mich., USA). The percent developer interference wasbelow 3% for each small molecule. The percent fluorophore interferencewas below 9% for each small molecule. The acceptable interference valueswere ≦10%.

m. Statistical Analyses

Three or more independent experiments were performed for each techniqueunless otherwise noted and the standard error of the mean (SEM)calculated and graphed. A one-tailed Student's t-test was used tocalculate p-values. P-values≦0.05 were considered significant.

ii) Activation of Stress Proteosome

Many small molecules are under investigation as potential therapeuticagents for a spectrum of Mendelian and complex genetic disorders.Screens for small molecules typically target a specific cellular pathwayrelated to, or implicated in, a particular disorder. However, varioussmall molecules with different known mechanisms of action, includinghistone deacetylase inhibitors (HDACi) and those that do not inhibithistone deacetylases, produce similar favorable outcomes in a widevariety of heterogeneous disease models in which different classes ofproteins, different cell types, and different molecular pathways areaffected. (Table 1).

TABLE 1 Similar Therapeutic Responses of Diverse Small Molecules inVarious Disease models. Disease Model Small Studied Molecule TherapeuticEffect References Huntington's 4PBA, TSA reduce neurodegenerationGardian et al., 2005 disease increase survival Oliveira et al., 2006Alzheimer's 4PBA, SFN reduce β-amyloid toxicity Park et al., 2009disease prevent neuronal cell death Ricobaraza et al., 2009 Diabetes4PBA, TSA, hyperglycemia normalization Ozcan et al., 2006 SFN improveglucose utilization Xue et al., 2008 Sun et al., 2008 Sickle Cell 4PBA,HU, increase F-cell production Hsiao et al., 2006 Disease TSA X-linked4PBA, TSA decrease very long chain McGuiness et al., 2003Adrenoleukodystrophy fatty acid levels Spinal Muscular 4PBA, HU,increase percentage of full Lunn et al., 2008 Atrophy TSA length SMN2transcript Fragile X 4PBA, HU, increase transcription of Gorski et al.,1985 Mental TSA FMR1 Chiurazzi et al., 1999 Retardation Ischemia 4PBA,SFN protection via antioxidant Qi et al., 2004 pathway Yoon et al., 2008Cystic Fibrosis 4PBA, TSA increase proper protein Choo-Kang et al., 2001trafficking Hutt et al., 2010 4PBA = 4-phenylbutyrate HU = hydroxyureaSFN = sulforaphane TSA = trichostatin A

The class I and class II HDACi, 4-phenylbutyrate (4PBA) and trichostatinA (TSA), have been extensively studied. In some cases, the beneficialeffect of HDACi treatment was attributed to an increase in theexpression of genes related to, or compensating for, the primarydisease-causing gene (Kemp et al., 1998; Gardian et al., 2005).

Studies of X-linked adrenoleukodystrophy (XALD), a neurologicaldisorder, indicate that the observed therapeutic overlap of HDACi andother small molecules is due to modulation of general cellular functionsrather than direct targeting of compensatory targets. Both 4PBA and TSAtreatment normalized the abnormally high levels of very long chain fattyacids (VLCFA) in human XALD fibroblasts and the ALD (Abcd1-/Y) mousemodel in vivo (McGuinness et al., 2003). 4PBA, but not TSA, increasedexpression of ABCD2, whose function overlaps that of the defectiveperoxisomal gene ABCD1 (Li et al., 2002). Thus, induction of ABCD2 doesnot directly correlate to the reduction of VLCFA levels in allinstances. However, 4PBA treatment of XALD fibroblasts also increasedmitochondrial and peroxisomal biogenesis, which are organelles requiredfor cellular detoxification and stress-sensing (Kemp et al., 1998;McGuinness et al., 2003). The induction of peroxisome proliferation by4PBA treatment is dependent on peroxisome biogenesis factor 11 alpha(PEX11α) (Li et al., 2002). PEX11β is required for constitutiveperoxisome abundance, whereas PEX11α is not. Rather, PEX11α inducesperoxisome proliferation in response to external stimuli or stress(Schrader et al., 1998). Therefore, the induction of the generalizedcellular stress response, also known as the adaptive cell survivalresponse, by small molecules was monitored (Kultz 2003). The therapeuticrelevance of modulating a general cellular response can explain howsmall molecules are able to elicit the observed diverse effects in abroad spectrum of diseases (e.g., Table 1, in which the amenablediseases exhibit minor defects at the cellular level regardless of theseverity of the clinical phenotypes).

The cellular stress response, which has been conserved from archaea toeukaryotes, protects against damage and promotes viability by adaptingcells to their environment (Kultz 2003). This response modulates theactivity of molecular chaperones and proteins (i) that affectreduction-oxidation regulation, (ii) that sense and repair DNA damage,(iii) that are involved in protein degradation, and (iv) that areinvolved in fatty acid, lipid, and energy metabolism. Stimulation ofthese adaptive survival pathways readjusts the cell to various stressorsand restores cellular homeostasis by inducing the heat shock response(HSR), the unfolded protein response (UPR), the autophagic response, theantioxidant response, and mitochondrial and peroxisomal biogenesis.Activation of the UPR and autophagy, proteostatic components of thestress response, and modulation of mitochondrial energetics canalleviate symptoms of neurodegenerative and aging disorders and extendthe lifespan of model organisms (Ong et al., 2010; Powers et al., 2009;Durieux et al., 2011). The present experiments indicate that jointactivation of the stress proteome and the subsequent reestablishment ofhomeostasis is beneficial to a broad range of diseases (Table 1).

The potential of four small molecules with overlapping therapeuticbenefits but different known functions (see Table 1), to induce theadaptive cell survival response was evaluated. The primary known modesof action of these small molecules: 4PBA, TSA, hydroxyurea (HU), andsulforaphane (SFN) are listed in Table 2. These studies show that, atconcentrations minimally affecting cellular proliferation, these fourdrugs increase mitochondrial biogenesis and peroxisome proliferation inboth normal and XALD human fibroblasts as well as in human K562erythroleukemic cells. These four pharmacological small moleculesinduced primary pathways that constitute the cytoprotective stressproteome. Thus, the therapeutic effects of these pharmacological smallmolecules or agents result from stimulation of the adaptive cellsurvival response and the reestablishment of cellular homeostasis.Identification of these common cellular responses allows screening formore clinically efficacious molecules, expands the repertoire ofcurrently treatable diseases to include those with unknown geneticetiology, and shortens the time to treatment for some diseases.

TABLE 2 Small Molecules and Mode of Action Small Molecule Known Mode ofAction Used Clinically 4-phenylbutyrate histone deacetylase inhibitorYes, Orally trichostatin A histone deacetylase inhibitor No hydroxyurearibonucleotide reductase inhibitor Yes, Orally sulforaphane phase IIdetoxification enzyme Yes, Clinical inducer Trials (Oral)

a. Small Molecule Induction of Mitochondrial Biogenesis and PeroxisomeProliferation

4PBA treatment of fibroblasts from healthy individuals, XALD patients,and patients with peroxisomal biogenesis disorders increasedmitochondrial mass and peroxisome proliferation 2- to 3-fold (Kemp etal., 1998; McGuinness et al., 2003; Wei et al., 2000). Other smallmolecules, including HU, TSA, and SFN, exhibit therapeutic overlap with4PBA (Table 1). To broaden the examination of small molecule inductionof mitochondrial biogenesis and peroxisome proliferation, normal humanfibroblasts were treated for four to five days with 4PBA, HU, TSA, orSFN. Drug concentrations were titrated to allow 100% viability andminimally affect cellular proliferation. Both mitochondrial biogenesisand peroxisomal biogenesis were monitored. Immunofluorescence stainingfor the mitochondrial membrane protein ATP synthase beta subunit (ATP5B)and the 70 kD peroxisomal membrane protein (ABCD3) revealed drug-inducedincreases in mitochondrial biogenesis and peroxisome proliferation,respectively, by all four small molecules or agents (FIG. 1A). FIG. 1Ashows immunofluorescence staining for mitochondrial membrane proteinATP5B (top) and the peroxisomal membrane protein ABCD3 (bottom). Normalhuman fibroblasts were treated with each drug for four to five days.(400× magnification). This identified mitochondrial biogenesis andperoxisome proliferation as a common cellular response to treatment withthese small molecules.

b. Beneficial Effects of 4PBA in XALD Cells Require IncreasedMitochondrial Function

4PBA-induced peroxisomal VLCFA β-oxidation (degradation) is dependent onmitochondrial long chain fatty acid (LCFA) β-oxidation (McGuinness etal., 2003). To determine whether 4PBA-induced peroxisomal VLCFAβ-oxidation was dependent on mitochondrial function, mitochondrialfunction was chemically inhibited with antimycin A (AA). Antimycin A(AA) is a cytochrome c reductase inhibitor that inhibits themitochondrial electron transport chain, mitochondrial biogenesis, andthus, cellular respiration (Ranganathan et al., 2009). The concentrationof AA, which was titrated to minimally affect basal levels of LCFA andVLCFA β-oxidation, did not exhibit any observable cellular toxicity, anddid not affect cellular proliferation. Treatment of XALD fibroblastswith 4PBA (1) induced LCFA and VLCFA β-oxidation (FIG. 1B) and (2)increased carbon dioxide release (Heinzer et al., 2003). FIG. 1B showsthe VLCFA analysis. XALD fibroblasts were treated with 4PBA in thepresence or absence of antimycin A (AA), a cytochrome c reductaseinhibitor, for five days and β-oxidation levels of LCFA (C16:0) andVLCFA (C24:0) levels were measured. However, in the presence of AA, the4PBA-induction of mitochondrial LCFA β-oxidation was inhibited. Thisinhibition concomitantly blocked the induction of peroxisomal VLCFAβ-oxidation. Thus, pharmacological reduction in peroxisomal VLCFA levelsby 4PBA treatment was not only dependent on mitochondrial LCFAβ-oxidation, but was more generally dependent on increased mitochondrialenergy production.

c. Beneficial Effects of Sodium Butyrate and Hydroxyurea in aB-Hemoglobinopathy Model Require Induced Mitochondrial Biogenesis

The clinical severity of β-hemoglobinopathies, such as sickle celldisease (SCD) and β-thalasemmia, was ameliorated by increasing thenumber of HbF-containing cells (F-cells). The increase in the number ofHbF-containing cells elevated the total HbF levels (Perrine 2008).Sodium butyrate (SB, an analog of 4PBA) and HU increased HbF levels inseveral contexts: in SCD and β-thalasemmia patients, in K562 cells, andin CD34+ derived hematopoietic stem cells (Keefer et al., 2006). Tofurther assess the pharmacological commonality of the induction ofmitochondrial and peroxisomal biogenesis in a disease model unrelated tothe neurological disease XALD, these responses were examined in K562cells. K562 cells are an erythroleukemic cell line that produces HbF andis commonly used as a model of the induction of F-cell production.

K562 cells were treated with SB and HU for four to ten days. Flowcytometric analyses demonstrated that both SB and HU significantlyincreased mitochondrial mass, the number of HbF-producing cells, andperoxisomal proliferation (FIGS. 1C, 1D, and 1E). FIGS. 1C, 1D, and 1Eshows results from the treatment of K562 cells with SB, HU, or PBS as acontrol (CON). (n≧3 unless otherwise noted).

To determine whether the induction of mitochondrial biogenesis by SB orHU treatment was necessary for the induction of HbF-producing cellproduction, K562 cells were treated with SB or HU in the presence orabsence of AA. The concentration of AA, which was again titrated tominimally affect basal levels of HbF-producing cells, did not exhibitany observable cellular toxicity and did not affect cellularproliferation. The induction of mitochondrial biogenesis and theinduction of HbF-producing cells by SB or HU treatment weresignificantly inhibited by AA (FIG. 1C-1D). FIG. 1C shows flowcytometric analysis of mitochondrial mass. After two to four days ofdrug treatment with or without AA, mitochondrial mass was measured usingMitotracker, a mitochondrial stain. Fold change (drug treated/control)is shown. FIG. 1D shows the analysis of HbF-producing cell production.After four days of drug treatment with or without AA, cells were stainedwith DAF. The relative number of cells producing HbF (DAF stained cells)were plotted. FIG. 1E shows flow cytometric analysis of peroxisomeproliferation. After eight to ten days of drug treatment, peroxisomeproliferation was measured using an antibody against the peroxisomalmembrane protein, PEX14 and a FITC-labeled secondary antibody. Foldchange (drug treated/control) is shown. In FIGS. 1C, 1D, and 1E, the *represents a statistically significant increase in a measurement betweendrug treated and control samples or a statistically significant decreasein a measurement between drug treated and drug treated samples in thepresence of AA (p≦0.05) (Bars=SEM). This finding indicates dependence ofthe beneficial effect (i.e., elevated HbF levels) on inducedmitochondrial biogenesis. Thus, the therapeutic effects observed in SCDmay involve the pharmacological induction of mitochondrial biogenesis bySB or HU.

d. Pharmacological Induction of Mitochondrial Biogenesis is Dependent onthe JNK Pathway

Mitochondrial biogenesis is stimulated by various signaling pathwaysthat activate the transcription factors peroxisomeproliferator-activated receptor gamma coactivator-1 alpha and beta (PGC1and PGC1β, respectively) (Lee et al., 2005). To determine whether theinduction of mitochondrial biogenesis by these small molecules requiredthe activation of a common kinase cascade or endothelial nitric oxidesynthase (eNOS), XALD fibroblasts were treated with (i) each inducingdrug and (ii) either an eNOS inhibitor or various specific kinaseinhibitors (Table 3).

TABLE 3 Kinase and endothelial nitric oxide synthase inhibitors tested.Inhibits Chemical Tested Mitochondrial Target Inhibitor ConcentrationsBiogenesis? MAPKK1 PD98059 10-100 μM no MAPKK1 arctigenin 1-50 μM no p38MAPK SB203580 10-100 μM no AMPK compound C 1-10 μM no constitutive eNOSL-NAME 0.1-1 mM no JNK 1, 2, and 3 SP600125 5-50 μM yes MAPKK1 and 2U0126 10-50 μM no protein kinase C RO-21-8425 1-30 μM no PI3K LY29400210-50 μM no Mitogen activated protein kinase kinase 1 (MAPKK1) is anupstream activator of extracellular signal-regulated kinases 1 and 2(ERK1/2). Adenosine monophosphate protein kinase (AMPK). Endothelialnitric oxide (eNOS). c-jun N-terminal MAPK (JNK).Phosphatidylinositide-3- kinase (PI3K).

4PBA, HU, TSA, and SFN increased mitochondrial biogenesis in XALDfibroblasts (FIG. 2A. top row). Inhibition of pathways, including thep38 mitogen activated kinase (MAPK), the extracellular regulated kinase,the MAPK kinases 1 and 2, the adenosine monophosphate kinase, theprotein kinase C, the phosphoinositide-3-kinase, and eNOS pathways, hadno effect on drug-induced mitochondrial biogenesis. OnlySP600125-inhibition (Bennett et al., 2001) of the stress activatedprotein kinase (SAPK) pathway, otherwise known as the c-jun N-terminalkinase (INK) pathway, with reduced pharmacological induction ofmitochondrial biogenesis as demonstrated by immunofluorescence stainingin treated XALD and by quantitative in-cell western analyses normalhuman fibroblasts. (FIG. 2A-B). In FIGS. 2A-B, the JNK inhibitorSP600125 (10 μM+inh) was utilized. FIG. 2A shows immunofluorescencestaining for mitochondrial membrane protein ATP5B. XALD fibroblasts weretreated with each drug for four days with or without SP600125. (400×magnification). FIG. 2B shows quantification of pharmacologicalinduction of mitochondrial biogenesis. Normal human fibroblasts weretreated with each of the small four molecules with or without SP600125for six days. Quantitative in-cell western analyses were performed usinganti-ATP5B.

As indicated by an increase in ATP5B staining, 4PBA, HU, TSA, or SFNtreatment alone significantly increased mitochondrial mass. In normalhuman fibroblasts, mitochondrial mass significantly increased 2.4-foldwith 4PBA treatment, 1.8-fold with HU treatment, 2.5-fold with TSAtreatment, and 2.2-fold with SFN treatment when compared to untreatedcells. The mitochondrial mass of cells treated with each drug and theJNK inhibitor did not significantly differ from the mitochondrial massof untreated cells.

In K562 cells, the JNK inhibitor SP600125 (Bennett et al., 2001) alsoblocked HU-induced mitochondrial biogenesis and consequentlyHbF-producing cell production (FIG. 2C-D). FIGS. 2C-D shows inhibitionof mitochondrial biogenesis and HbF-containing cell production. K562cells were treated for four days with HU in the presence or absence ofSP600125 or PBS as a control (CON). FIG. 2C shows mitochondrial mass(plotted as in FIG. 1C) and FIG. 2D shows the relative number ofHbF-producing cells were determined by staining with Mitotracker andDAF, respectively (n=2). In fully competent hematopoietic stem cellsHU-stimulated F-cell production was similarly dependent on the JNKpathway. The JNK pathway is activated by external stressors and stimulisuch as heat shock, osmotic shock, and ultraviolet irradiation (Yang etal., 2003). Therefore, these data indicate that the same cytoprotectivepathway is involved in the pharmacological induction of mitochondrialbiogenesis by 4PBA, HU, TSA, and SFN treatment.

Activation of the JNK pathway led to activation of the transcriptionfactor JUN via phosphorylation. PGC1α and PGC1β maintained basal levelsof mitochondria. PGC1α also induced mitochondrial biogenesis underphysiological stress such as during adaptive thermogenesis, enduranceexercise, or fasting (Kultz 2005). JUN transcript levels weresignificantly increased after six hours of treatment with 4PBA, HU, orTSA. PGC1α and PGC1β transcript levels were significantly increasedafter 48 hours or more of treatment with each of the four smallmolecules (FIG. 2E). JUN transcript levels were not consistently changedafter SFN treatment. FIG. 2E shows mRNA expression of JUN andmitochondrial transcription factors PGC1α and PGC1β by RT-PCR. Therelative gene expression for each treatment compared to untreated normalhuman fibroblasts was calculated and the fold change (drugtreated/untreated) is shown. Measurements of PGC1α and PGC1β levelsafter SFN treatment were performed twice in duplicate.

However, JNK phosphorylation was increased within 24 hours of treatmentwith each drug, including SFN (FIG. 2F). Thus, the JNK pathway wasactivated and the abundance of the mitochondrial transcription factorswas increased following 4PBA, HU, TSA, or SFN treatment. FIG. 2F showsimmunoblot analysis of JNK phosphorylation (46 kDa). Normal humanfibroblasts were treated at various time points. Treatment was initiatedat the indicated times prior to collection. The average of the maximumprotein expression (within 24 hours) for three or more independentexperiments is shown as fold change (drug treated/untreated). Actin (43kDa) was the loading control. M denotes the marker lane. (0.05≦p≦0.10for 4PBA and TSA treated samples). The * statistically significantincrease in a measurement between drug treated and untreated or controlsamples or a statistically significant decrease in a measurement betweendrug treated and drug treated samples in the presence of SP600125(p≦0.05) (n≧3 independent experiments unless otherwise noted)(Bars=SEM).

e. Small Molecule Activation of the Adaptive Cell Survival Response

In three different cell types (normal human fibroblasts, XALDfibroblasts, and K562 cells) inhibition of the stress-activated JNKpathway blocked the small molecule induction of mitochondrialbiogenesis. Mitochondrial biogenesis is a necessary response for thereduction of VLCFA in XALD cells and for the increase in HbF levels inK562 cells. The mitochondria are involved in cellular adaptation tostress, indicating that a common cellular response to thesepharmacological small molecules or agents may be stimulation of thestress proteome. Expression of key components of the HSR, UPR, theautophagic response, and the antioxidant response were monitored at thetranscriptional, translational, and/or post-translational level. Theseresponses are known to be transiently activated in response to variouscellular stressors. The degree of induction of each component can varydepending on the metabolic state of the cell (e.g., confluency, cellpassage number) (Kultz 2003; Lallemand et al., 1998; Westerheide et al.,2009). Due to the transient nature of these responses, severalcomponents of each pathway were monitored at various time points withina 24 hour treatment. At least two normal human fibroblast lines weretested in three or more independent experiments. An increase in mRNAexpression after 2 hr., 6 hr., or 18 hrs. of treatment occurred. Theaverage of the maximal increase in protein expression within a 24 hourtime frame was also compared to the untreated sample. Activation of eachpathway was assessed by an increase in one or more components of eachpathway.

f. Small Molecule Activation of the Heat Shock Response

Induction of the HSR is a hallmark of the adaptive cell survivalresponse (Fedoroff 2006). To evaluate its activation, the expression ofheat shock protein (HSP) 40, HSP 70, and HSP 90 family members wasmonitored. 4PBA, TSA, or SFN increased transcription of the HSP 70 kDaprotein 1A (HSPA1A). All four small molecules or agents increasedtranscription of DNAJ HSP40 homolog subfamily C member 3 (DNAJC3) andtranscription of HSP 90 kDa class A member 1 (HSP90AA1; FIG. 3A). FIG.3A shows RT-PCR analyses of mRNA expression of HSR genes. Normal humanfibroblasts were treated and the mRNA expression of HSPA1A (HSP70),DNAJC3 (HSP40), and HSP90AA1 (HSP90) was measured and plotted as in FIG.2E. P-value for SFN DNAJC3 measurement is 0.10. HSPA1A transcriptexpression was not stimulated by HU treatment at the time pointsexamined.

However, total HSP70 and HSP90 protein levels were significantlyincreased with all four small molecules or agents (FIG. 3B). Thepharmacological induction of HSR mRNA and protein expression was similarto that caused by mild heat shock (Clark et al., 2009). Thus, the HSRwas activated by 4PBA, HU, TSA, or SFN treatment. FIG. 3B showsimmunoblot analyses of HSP expression. Normal human fibroblasts weretreated at various time points within 24 hours and the average of themaximum protein expression of HSP70 (70 kDa) and HSP90 (90 kDa) isplotted as in FIG. 2F. Actin (43 kDa) was used as a loading control.(UT=untreated cells; * statistical significance (p≦0.05); Bars=SEM forn≧3 independent experiments).

g. Small Molecule Activation of the Unfolded Protein Response

UPR markers evaluated included the central UPR regulator glucoseregulated protein 78 (BIP) and other components that are stimulatedafter the activation of the three ER transmembrane receptors: PKR-likeER kinase (PERK), inositol requiring 1 (IRE1), and activatingtranscription factor 6 (Fedoroff 2006). Elongation initiation factor 2alpha (eIF2a) is phosphorylated by PERK, attenuates general translation,and induces activating transcription factor 4 (ATF4). CHOP promotesreactivation of PERK. XBP1 mRNA is non-conventionally spliced by theendonuclease activity of IRE1 upon UPR activation. BIP expression wassignificantly increased at the transcriptional and translational levelsafter treatment with 4PBA, HU, TSA, or SFN (FIG. 4A-4B). Treatment withthese small molecules also increased ATF4 and CHOP mRNA expression (FIG.4A), modestly increased eIF2α phosphorylation (FIG. 4B), and increasedthe total amount of XBP1 protein and the amount of spliced XBP1 protein(FIG. 4C).

FIG. 4A shows RT-PCR analyses of mRNA expression of UPR genes. Normalhuman fibroblasts were treated with drugs as indicated. The expressionof the UPR genes ATF4, CHOP, and BIP was measured as described in FIG.2E. FIG. 4B shows immunoblot analyses of UPR protein expression. Normalhuman fibroblasts were treated at various time points and the average ofthe maximum protein expression of BIP (78 kDa) and phosphorylated eIF2α(38 kDa) is plotted as in FIG. 2F. Actin (43 kDa) was used as a loadingcontrol and is the same blot as FIG. 2F. UT denotes untreated cells. Mdenotes marker lane. FIG. 4C shows XBP1 splicing. Normal humanfibroblasts were treated. The expression of the unspliced form (XBP1-us;33 kDa) and the activated and spliced form of XBP1 (XBP1-s; 54 kDa) wasanalyzed by immunoblotting. BP1-s is a larger protein than XBP1-us dueto non-canonical mRNA splicing which results in a largercarboxy-terminal domain. The percentage of XBP1-s to XBP1-us increasedwith each treatment as shown (24 hr. time point). The average of themaximum expression of XBP1-s (within 24 hrs.) is plotted as in FIG. 2F.Actin was used as a loading control. (P=0.08 for the TSA treatedvalues; * statistical significance (p≦0.05); Bars=SEM for n≧3independent experiments).

Thus, the pro-survival capabilities of all three UPR branches wereactivated at the transcriptional, post-transcriptional, translational,and post-translational levels by treatment with these small molecules.

h. Small Molecule Activation of Autophagy

Adverse growth conditions increase the energetic demand on a cell, whichin turn, stimulates the catabolic processes of autophagy to promoteutilization of damaged and excess proteins and damaged organelles forcellular nutrients (Martinet et al., 2009). To monitor activation ofautophagy, the expression of three classical autophagy markers wasexamined. The three classical autophagy markers are: (i) beclin-1(BCN1), (ii) autophagy protein 5 (ATG5), and (iii)microtubule-associated protein 1 light chain 3 (LC3 or APG8).Autophagosome formation is signaled by the phosphorylation of BCN1 byJNK1 and is dependent on the conjugation of ATG5 and ATG12 and thecleavage (LC3-I) and phosphatidylethanolamine lipidation (LC3-II) ofAPG8. Treatment with 4PBA, TSA, or SFN increased BCN1 and ATG5 mRNAlevels (FIG. 5A). FIG. 5A shows RT-PCR analyses of mRNA expression ofautophagy genes. Normal human fibroblasts were treated as indicated. Theexpression of BCN1 and ATG5 were measured as described in FIG. 2E. AfterTSA treatment, the levels of ATG5 mRNA were measured in two independentexperiments in duplicate. The p-value for the ATG5 HU treated samples is0.10. The p-values for the BCN1 HU and SFN samples are 0.23 and 0.06,respectively.

After HU treatment, ATG5 mRNA levels also increased, but BCN1 mRNAlevels were not reproducibly changed within six hours of treatment.Treatment with each of these small molecules significantly increased theproportion of LC3-II to LC3-I, a hallmark of autophagy activation (FIG.5B). FIG. 5B shows immunoblot analyses of the cleavage and lipidation ofautophagy protein APG8. Normal human fibroblasts were treated for 4hrs.-24 hrs. and the average of the maximum proportion of APG8 LC-II (13kDa), the cleaved and lipidated form of APG8, to APG8 LC3-I (17 kDa) isplotted as in FIG. 2F. A 24 hour time point is shown. Actin (43 kDa) wasused as a loading control. UT denotes untreated cells. Therefore,treatment with each of the four small molecules activated the autophagypathway.

i. Small Molecule Activation of the Antioxidant Response

The antioxidant response detoxifies the cell and regulatesreduction-oxidation (redox) homeostasis by neutralizing the effects ofreactive oxygen species (ROS) and reactive nitrogen species (RNS). ROSand RNS are second messengers of the adaptive cell survival response(Fedoroff 2006). The expression of three key components was examined—(i)nuclear factor erythroid 2-like 2 (NFE2L2), (ii) heme oxygenase 1(HMOX1), and (iii) superoxide dismutase 2 (SOD2). NFE2L2, atranscription factor that binds the antioxidant response element, isinvolved in the chemoprotective response provided by SFN (Myzak et al.,2004). After treatment with 4PBA, HU, TSA, or SFN, NFE2L2 and SOD2 mRNAlevels increased (FIG. 5C). FIG. 5C shows RT-PCR analyses of mRNAexpression of antioxidant genes. Normal human fibroblasts were treatedfor 18 hours. The expression of NFE2L2, HMOX1, and SOD2 was measured asdescribed in FIG. 2E. The p-value for the NFE2L2 HU measurements was0.11.

HMOX1 mRNA levels significantly increased with HU, TSA, or SFN treatmentand significantly decreased with 4PBA treatment. SOD2 protein levelsalso significantly increased (FIG. 5D). FIG. 5D shows immunoblotanalyses of SOD2 protein expression. Normal human fibroblasts weretreated for two to five days. The relative expression of SOD2 (26 kDa)is plotted for each treatment compared to UT cells as in FIG. 2F. Actin(43 kDa) was used as a loading control. (*statistical significance(p≦0.05); Bars=SEM for n≧3 independent experiments unless otherwisenoted). Thus, cellular antioxidant defense mechanisms were induced atthe transcriptional and translational levels by treatment with each ofthese small molecules.

j. HU and SFN do not Inhibit Class I and II HDACs

These small molecules activate the stress proteome and share similarcellular responses in diverse disease models. Two of the smallmolecules, 4PBA and TSA, directly inhibit histone deacetylase activityin vitro (Jung 2001). Whether inhibition of class I and class II HDACactivities is a shared biochemical function that could account for theeffects of all four small molecules was examined. Using lysates fromHeLa cells and primary human fibroblasts, an in vitro colorimetric assayof HDAC activity was performed. Two concentrations of each smallmolecule were used. The first concentration was that used to treatprimary human fibroblasts in the experiments reported herein, whichminimally affects growth. The second concentration was ten times thefirst concentration and is lethal in cell culture. 4PBA and TSAsignificantly decreased HDAC activity when compared to untreated HeLaand human fibroblast lysates (FIG. 6A-B). However, neither concentrationof HU nor SFN reduced class I or II HDAC activity in lysates. In FIG. 6,HDAC activity was measured using extracts from two cell lines as shown.(*p≦0.00009; paired t-test; Bars=SEM for n≧3 independent experiments).

A physiological increase in histone acetylation after SFN treatment ofwhole cells that may reflect a change in gene expression that is not theresult of direct biochemical inhibition of HDAC enzymatic activity bySFN was demonstrated [Myzak et al., 2004]. While the inhibition of HDACactivity by 4PBA or TSA treatment may induce the stress proteome, HU andSFN induce the stress proteome independent of direct HDAC inhibition.

k. Beneficial Effects of SFN in Spinal Muscular Atrophy Cells areDependent on the JNK Pathway, Autophagy, Mitochondrial Biogenesis andSIRT1 Activity

To determine which of the drug-induced stress pathways are necessary forthe therapeutic effects of these small molecules, their effects inspinal muscular atrophy fibroblasts were examined. Ninety-five percentof spinal muscular atrophy patients have a homozygous deletion of thetelomeric SMN1 (chr 5q13) gene or gene conversion at exon 7 or 8(Lefebvre et al., 1995). However, patients have one or more copies of acentromeric SMN1 pseudogene, SMN2. Compared with SMN1, SMN2 has a C to Ttransition within an exonic splice enhancer that results in the skippingof exon 7 (SMND7) and an unstable protein that is degraded (Sumner etal., 2003; Lorson et al., 1998). Only 15-30% of SMN2 transcripts includeexon 7 and are full length (FL-SMN). Since the clinical severity ofspinal muscular atrophy patients inversely correlates with the levels ofFL-SMN transcript and SMN protein, therapeutic strategies that increaseFL-SMN and SMN protein production offer promise.

If these three small molecules and SFN share a common therapeuticmechanism, SFN treatment should also increase FL-SMN mRNA and SMNprotein expression. To determine whether a common effect of treatmentwith these four small molecules is the induction of the stress proteome,the following were examined: (i) the induction of mitochondrialbiogenesis by these four small molecules in spinal muscular atrophyfibroblasts, (ii) the effect of SFN treatment on the expression ofFL-SMN mRNA and SMN protein in spinal muscular atrophy, and (iii) theability of inhibitors of the various stress pathways to block inductionof FL-SMN mRNA and SMN protein expression.

Similar to treatment of X-linked adrenoleukodystrophy fibroblasts, K562cells and normal human fibroblasts, treatment of spinal muscular atrophyfibroblasts with each of the four small molecules increasedmitochondrial biogenesis as monitored by Mitotracker staining, acell-permeant mitochondrion-selective dye (FIG. 7). FIG. 7 shows thatmitochondrial biogenesis is induced in spinal muscular atrophyfibroblasts by 4PBA, HU, TSA or SFN treatment Immunofluorescencestaining for mitochondria using Mitotracker Red CMXROS. Spinal muscularatrophy fibroblasts were treated with 2.5 mM 4PBA, 300 mM HU, 100 nM TSAor 2.5 mM SFN for 5 days (×80 magnification).

SFN treatment significantly increased FL-SMN mRNA expression comparedwith total SMN transcription (FL-SMN plus SMND7) in two spinal muscularatrophy type I cell lines (GM09677 two copies SMN2 and GM00232 one copySMN2) and one spinal muscular atrophy type III cell line (96-2906 fourcopies SMN2; FIG. 8A) (Sumner et al., 2003). Cell lines with greaternumbers of SMN2 gene copies respond better to treatment.

FIG. 8 shows the induction of FL-SMN mRNA expression and SMN proteinexpression by SFN is dependent on the JNK pathway, autophagy,mitochondrial biogenesis and SIRT1 activity. For example, FIG. 8A showsthe RT-PCR analyses of FL-SMN mRNA expression compared with total SMNtranscript expression. Type I and type III spinal muscular atrophy (SMAI or SMA III) fibroblasts were treated with SFN for the indicated times.The relative fold increase in the ratio of FL-SMN/total SMN transcriptscompared with the untreated cell line (normalized to 1) is plotted. Celllines from left to right are GM09677, GM00232 and 2906. N≧2 independentexperiments.

FIG. 8B shows RT-PCR analyses of FL-SMN and total SMN mRNA expression.Spinal muscular atrophy fibroblasts were treated with 1.5 mM SFN alonefor 51 hr. (SMA I, GM09677) or treated with 0.5 mM SFN for 8 hr. (SMAIII, 2906) with or without 12.5 mM SP600125 (JNK inhibitor), 2.5 mM3-methyladenine (autophagy inhibitor, APG), 5 mg/mL antimycin A(mitochondrial inhibitor, MITO) or 3 mM sirtinol (SIRT1 inhibitor). Thefold increases in FL-SMN expression compared with either GAPDH or totalSMN expression and the fold increase in total SMN expression comparedwith GAPDH expression after SFN treatment is plotted. Untreated valueswere normalized to 1 and are indicated by the horizontal line. (n=1 percell line, performed in duplicate)

FIG. 8C shows immunoblot analyses of SMN protein expression. Spinalmuscular atrophy type I (SMA I; GM09677, n=7) or type III (SMA III;2906, n=2) fibroblasts were treated with either 1.5 mM SFN or 0.5-2 mMSFN, respectively, for 48-72 hr. The average of the relative expressionof SMN protein (39 kDa) is plotted. Actin (43 kDa) was used as a loadingcontrol.

FIG. 8D shows a summary of the effects of stress pathway inhibitors onSMN protein expression. Spinal muscular atrophy type I (SMA I; GM09677)fibroblasts were treated with either 300 mM HU (n=1) or 1.5 mM SFN (n≧3)in the presence or absence of 10 mM SP600125 (INK inhibitor), 1 mM3-methyladenine (APG inhibitor), 2-4 mg/ml antimycin A (MITO inhibitor)or 2 mM sirtinol (SIRT1 inhibitor) for 48 h. SMN protein expression wasmeasured by quantitative immunoblot analyses using actin as a loadingcontrol. Fold increases in SMN protein expression are plotted andrepresented numerically in the table below the graph. Untreated valuesare normalized to 1. (*P≦0.05; bars=SEM).

After SFN treatment, the relative expression of FL-SMN transcriptsincreased when normalized to either GAPDH transcript levels or to totalSMN transcript levels (FIG. 8B). However, total SMN transcript levelsremained unchanged when normalized to GAPDH transcript levels aftertreatment. Therefore, SFN treatment increases the quantity of FL-SMNtranscripts by enhancing the inclusion of exon 7 rather than increasingoverall transcriptional expression from the SMN2 gene.

The reduction in very long-chain fatty acid levels in X-linkedadrenoleukodystrophy fibroblasts by 4PBA was dependent on the inductionof mitochondrial biogenesis and that the induction of HbF production inK562 cells by HU was dependent on the JNK pathway and mitochondrialfunction (FIGS. 1B and 1D and FIG. 2D). To determine which stresspathways are necessary for the increase in FL-SMN production, spinalmuscular atrophy fibroblasts were co-treated with SFN and eitherSP600125, an inhibitor of the JNK pathway (Bennett et al., 2001),3-methyladenine, an inhibitor of the autophagy pathway (Seglen et al.,1982), antimycin A, a mitochondrial inhibitor (Ranganathan et al.,2009), or sirtinol, an inhibitor of SIRT1 activity (Grozinger et al.,2001). The concentration of each inhibitor did not affect cell growth orviability. SIRT1, an NAD+-dependent deacetylase, is known to regulatecellular stress responses, cellular metabolism and cellular survival(Salminen et al., 2009). Specifically, SIRT1 inhibition reduces theinduction of HSR genes (Westerheide et al., 2009); SIRT1 activityenhances SOD2 expression (Anastasiou et al., 2006); SIRT1 negativelyregulates the mammalian target of rapamycin triggering autophagy (Haigiset al., 2010); and SIRT1 activates mitochondrial biogenesis via thestress-responsive transcription factor PGC1a (Yu et al., 2009). Theyeast ortholog Sir2 is necessary for the induction of mitochondrialbiogenesis by 4PBA or HU in Saccharomyces cerevisiae (Cha 2010). Therewere no marked changes in SIRT1 protein expression after treatment ofnormal human fibroblasts with 4PBA, HU, TSA or SFN. However, SIRT1 canbe activated by various post-translational changes. Therefore, thepotential involvement of SIRT1 in the therapeutic effects of these drugsby inhibition studies was evaluated. Biochemically blocking the JNKpathway, the autophagy pathway, induction of mitochondrial biogenesisand SIRT1 activity prevented the increase in FL-SMN transcript levels inSFN-treated spinal muscular atrophy fibroblasts indicating the necessityand potential concerted action of each of these pathways for thetherapeutic response to SFN treatment (FIG. 8B).

To further demonstrate that induction of these various stress pathwaysis necessary for the therapeutic effects of the small molecules understudy, the expression of total SMN protein expression after either SFNor HU treatment in the presence or absence of the stress pathwayinhibitors was monitored. After 48-72 hr. of treatment, SFN increasedSMN protein expression 1.57-fold in spinal muscular atrophy type Ifibroblasts and 1.64-fold in spinal muscular atrophy type IIIfibroblasts (FIG. 8C). In separate experiments, HU or SFN treatmentincreased SMN protein expression 1.4- and 1.46-fold, respectively (FIG.8D). The pharmacologically induced increase in SMN protein expressionwas significantly reduced by the various stress pathway inhibitors,indicating that the increase in SMN protein expression by both HU andSFN was dependent on activation of the JNK pathway, autophagy,mitochondrial biogenesis and SIRT1 (FIG. 8D). At the concentrationsused, the stress pathway inhibitors had no effect on basal levels of SMNprotein expression. The magnitude of the increases in FL-SMN expressionand SMN protein expression in SFN-treated spinal muscular atrophy cellscompared with untreated cells is similar to that observed after 4PBA, HUor TSA treatment in SMA fibroblasts (Andreassi et al., 2004; Grzeschiket al., 2005; Avila et al., 2007). Since SFN induction of FL-SMNtranscript expression and SMN protein expression is dependent on the JNKpathway, autophagy pathway, mitochondrial biogenesis and SIRT1, thetherapeutic potential of the activation of the stress proteome mayrequire the concerted action of each of the individual stress pathways.

l. SIRT1 Activation is not a Common Mechanism of Action of 4PBA, HU, TSAand SFN

To determine whether SIRT1 activation was a common biochemical activityof the small molecules under study, a fluorescence-based in vitro SIRT1activity assay was performed with two concentrations of each drug: thatused to treat normal primary human fibroblasts in the experimentsreported here (low), and 10 times that concentration, which is lethal incell culture (high). HU and TSA had no effect on SIRT1 activity, whereas4PBA (50 mM; high) and SFN (5 and 50 mM) significantly inhibited SIRT1activity by 54, 14 and 40%, respectively (FIG. 9). This was anon-cell-based assay, and therefore, it is difficult to determine theeffects of 4PBA and SFN on SIRT1 activity in vivo. Regardless, whileSIRT1 activation may play a role in the induction of cellular stressresponses (Anastasiou et al., 2006), direct biochemical activation orinhibition of SIRT1 is not necessary for the common pharmacologicalinduction of the adaptive cell survival response by all four smallmolecules.

FIG. 9 shows that biochemical SIRT1 activation is not necessary for theinduction of the stress proteome by 4PBA, HU, TSA and SFN. SIRT1activity assay. SIRT1 activity was measured by incubating an acetylatedlysine substrate with human recombinant SIRT1, cosubstrate NAD+ and theindicated concentration of each small molecule. The percent inhibitionof SIRT1 is plotted. (Bars=SEM for triplicate measurements; *p≦0.05;paired t-test).

Collectively, the data presented herein demonstrate that pharmacologicalsmall molecules or agents without known overlapping molecular functionsinduce mitochondrial and peroxisomal biogenesis and the transcriptionand translation of signature components of the adaptive cell survivalresponse (HSR, UPR, autophagy, and antioxidant response). Under adversecellular conditions, mitochondria induce either the endogenous adaptivecell survival response reestablishing homeostasis (mild stress) orapoptosis (severe stress) (Lee et al., 2005). The small moleculesstudied herein exhibited a biphasic or hormetic dose response (Calabreseet al., 2010). At the low doses used herein, the four small moleculesactivated the stress proteome and produced beneficial effects; and athigher doses, the four small molecules were cytotoxic. Thepharmacological induction of mitochondrial biogenesis is consistent withthe significant increase in energy expenditure required for metabolicadaptation to increased cellular stress.

The results presented herein demonstrate the therapeutic importance ofthe pharmacological induction of mitochondrial biogenesis in XALD cellsand K562 cells, a model of HbF induction. The reduction in VLCFA levelsin 4PBA-treated XALD fibroblasts and the increase in HbF-producing cellnumber in HU-treated K562 cells depended on the stress-activated JNKsignaling cascade and the subsequent induction of mitochondrialbiogenesis. Since the small molecules have different known functions(Table 2) and different chemical structures, yet elicited similarmolecular responses, these data demonstrate generally that smallmolecules with varying functions can activate the adaptive cell survivalresponse and elicit similar therapeutic responses.

Beneficial therapeutic effects that improve disease pathology and thatare independent of altering the specific genetic mutation have been welldocumented. Examples include TSA treatment of Duchenne's musculardystrophy models, losartan treatment of Marfan's syndrome patients, and4PBA, HU, or TSA treatment of spinal muscular atrophy models (Lunn etal., 2008; Minetti et al., 2006; Habashi et al., 2006). Such examplesprovide an elegant illustration of corrective biological responses fromindirect interventions.

A therapeutic role for adaptive survival pathways has been observed in arange of diseases. Overexpression or pharmacological induction of heatshock proteins (i) corrected the defect in Niemann-Pick patient celllines, a lysosomal storage disorder, (ii) ameliorated the phenotype ofspinal bulbar muscular atrophy (SBMA) mouse models, and (iii) reducedprotein aggregation in studies of Huntington's disease, a neurologicaldisorder (Evans et al., 2010). Induction of the UPR proteins XBP1 andATF6 protected against ischemia-reperfusion injury (Toth et al., 2007).Pharmacological induction of autophagy or overexpression of autophagyproteins protected against ischemia-reperfusion injury, improved mutantprotein clearance, and reduced protein toxicity in cell and animalmodels of Huntington's disease, Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia,and SBMA (Martinet et al., 2009; Madeo et al., 2009). Antioxidant and4PBA treatment improved insulin sensitivity and glucose homeostasis indiabetic mouse models (Ozcan et al., 2006; Liu et al., 2009).4PBA-induced mitochondrial biogenesis can help to normalize glucoselevels because mitochondrial dysfunction is a key contributor to insulinresistance. 4PBA-induced expression of SOD2 increases neuroprotection inALS (Petri et al., 2006). Improved mitochondrial membrane potential andincreased peroxisome proliferation induced by 4PBA treatment promotedneuronal integrity in Alzheimer's studies (Santos et al., 2005).Although 4PBA, HU, TSA, and SFN treatment induced all four adaptivesurvival pathways examined, the induction of all pathways may not benecessary for a therapeutic outcome in every responsive disease.

The induction of the adaptive cell survival response and the subsequentreestablishment of cellular homeostasis explain why these diversepharmacological small molecules or agents produce similar therapeuticeffects in such a variety of disease models (Table 1). The diseasesresponsive to these small molecules have mild cellular abnormalities,i.e., the cells are viable even though their suboptimal function maylead to severe clinical manifestations. The pharmacological enhancementof the adaptive cell survival response and the subsequentreestablishment of cellular homeostasis beneficially altersdisease-related metabolic stress, promotes cell viability, andameliorates some mild cellular genetic abnormalities without directlytargeting the disease-causing gene. For example, enhancement ofautophagy in the SOD1 transgenic ALS mouse model cleared proteinaggregates and significantly increased lifespan (Madeo et al., 2009).Also, TSA treatment of Abcd1−/− fibroblasts normalized β-oxidationlevels of VLCFA independent of Abcd1 or Abcd2 expression (McGuiness etal., 2003). This indirect approach to therapy offers the advantage ofpharmacologically modulating the adaptive capacity of the endogenouscellular machinery, specifically the stress proteome and mitochondrialfunction, to compensate for metabolic abnormalities and improve cellularfunction. This provides a rationale for the treatment of diseases whosespecific genetic abnormalities are unknown, including many complexdisorders, and may shorten the time to treatment for some currentlyuntreatable diseases.

The four small molecules disclosed herein can activate the stressproteome via their known biochemical activities or via uncharacterizedmolecular interactions within cells. For example, in K562 cells, theproduction of ROS increases F-cell production after SB and TSAtreatment, but not after HU treatment (Hsiao et al., 2006). This resultindicates that F-cell production is stimulated by differing pathways.Identification of the cellular responses necessary for downstreamtherapeutic effects of interest, i.e., mitochondrial biogenesis, canassist screening for small molecules with optimal clinical effects.

The small molecules investigated here were identified by effects ondisease outcomes. Establishment of the therapeutic potential of theadaptive cell survival response by these small molecules providestargets for the identification of more efficacious small molecules withlower toxicity. The exploitation of the innate cellular survival programmay ameliorate disease symptoms in a spectrum of disorders with mildcellular phenotypes without targeting a specific molecule or signalingpathway for each individual disease. The amenable disorders not onlyinclude those with mitochondrial or peroxisomal defects, increasedoxidative stress, or protein conformation or trafficking defects, butalso include aging disorders and complex diseases with unknown geneticetiology and mild cellular phenotypes. Therapy that targets homeostaticregulation could have a profound effect on medicine.

iii) Stimulation of HSP by Sulforaphane

Sulforaphane stimulated HSP and mitochondrial biogenesis in severalgenetic disorders. The data provided herein demonstrated thatsulforaphane induces known cellular stress response pathways and heatshock proteins that protect cells from mild metabolic disturbances. Thestress proteome response to low doses of sulforaphane includes effectson redox regulation, DNA damage sensing and repair, molecularchaperones, fatty acid and lipid metabolism and energy metabolism.

Sulforaphane and related HDAC and non-HDAC inhibitors are hormetic drugsthat induce a general “cell-protective” response, as demonstrated in anumber of neurological diseases. The cellular repair effects contrastsharply with the cellular and genetic damage reported to occur followingexposure to high doses of the same drugs.

Sulforaphane induces known cellular stress response pathways and heatshock proteins that protect cells from mild metabolic disturbances. Thestress proteome response to low doses of sulforaphane includes effectson redox regulation, DNA damage sensing and repair, molecularchaperones, fatty acid and lipid metabolism and energy metabolism(Keefer et al., 2006). The data presented herein shows the effects ofsulforaphane on the heat shock response. mRNA expression of heat-shockproteins genes was markedly increased (3-7-fold) in normal humanfibroblasts following exposure to 5 micromolar (μM) sulforaphane. Theheat shock proteins affected are: HSP40, USP70, and USP90.

Heat shock proteins and ubiquitin/26 S proteasome subunits were inducedin the liver of mice at time points that were 3- and 12-hours aftertreatment with a single dose of sulforaphane (90 mg/kg) by gavage (Hu etal., 2005). In HeLa cells and COS1 cells, sulforaphane enhancedproteasomal activity. Sulforaphane also increased the gene expression ofHsp27 by activating heat shock factor 1 (HSF1), which is the majortranscription factor that regulates the expression of heat shockproteins (Gan et al., 2010, which is incorporated herein in its entiretyby reference). In HeLa cells, sulforaphane treatment caused dissociationof HSF1 from its negative regulators Hsp90 and Hsp70, followed bynuclear translocation of the transcription factor. In a luciferasereporter system, sulforaphane activated HSF1-mediated transcription ofthe reporter, and the expression of the endogenous HSF1-target genesHsp70 and Hsp27.

The mechanism of HSF1 activation by sulforaphane is presently unknown.However, HSF1 is activated by electrophiles, such as the quinone methidecelastrol, hydrogen peroxide, menadione, arsenic trioxide,15-deoxy-PGD2, and 4-hydroxynonenal. It is possible that sulforaphanemodifies directly specific cysteine residues of HSF1 thus altering thebinding of HSF1 to its negative regulators Hsp90 and Hsp70 and/or theability of the transcription factor to trimerize and bind to DNA.Another interesting possibility is that sulforaphane, through itsability to inhibit the activity of the cytoplasmic non-histone proteindeacetylase HDAC6, and consequently enhance Hsp90 acetylation (Gibbs etal., 2009), may inhibit its association with HSF1.

iv) In Vitro Sulforaphane Studies

As described herein, in vitro studies demonstrated that sulforaphane(SF) stimulated the metabolic pathways that comprise the adaptivecellular stress response. Mitochondrial and peroxisomal biogenesis wereincreased 2- to 3-fold as compared to untreated cells. In addition, theJNK pathway was activated and the concentration of mitochondrialtranscription factors was increased 2- to 4-fold.

Heat Shock Response (HSR) induction is a hallmark of the adaptive cellsurvival response. Sulforaphane (SF) increased the transcription ofHSP70 by 7-fold and increased the transcription of both HSP40 and HSP 90by 3-fold. SF increased the total HSP70 and HSP90 protein levels by 3 to4-fold. The pharmacological induction of HSR mRNA and protein expressionwas similar to that caused by mild heat shock. Sulforaphane treatmentactivated the pro-survival capabilities of all three branches of the UPRat the transcriptional, post-transcriptional, translational, andpost-translational levels. The central UPR regulator glucose regulatedprotein 78 (BIP) was increased 2-3 fold at the transcriptional andtranslational levels. SF treatment also increased ATF4 and CHOP mRNAexpression 4-5 fold, modestly increased eIF2a phosphorylation, andincreased the total amount of XBP1 protein and the amount of splicedXBP1 protein 3-4.

The expression of three classical autophagy markers, beclin-1 (BCN1),autophagy protein 5 (ATG5), and microtubule-associated protein 1 lightchain 3 (LC3 or APG8), was examined after SF treatment. BCN1 and ATG5mRNA levels increased 5-fold and 3-fold, respectively, and theproportion of LC3-II to LC3-I, a hallmark of autophagy activation, wassignificantly increased. The cellular antioxidant defense mechanismswere induced at the transcriptional and translational levels. Theexpression of three key components was also increased: nuclear factorerythroid 2-like 2 (NFE2L2) by 3-fold, hemeoxygenase 1 (HMOX1) by6-fold, and superoxide dismutase 2 (SOD2) by 4-fold. NFE2L2, atranscription factor that binds the antioxidant response element, isknown to be involved in the chemoprotective response provided bysulforaphane.

v) Effect of Fever, Cellular Stress, and Hydroxyurea in Autism

Autism is a clinically heterogeneous disorder, which results from avariety of nonlethal genetic disorders and epigenetic effects thataffect related metabolic pathways. Some of these pathways respond topharmacological stimulation of cellular stress responses. The geneticand environmental factors underlying its various forms are the focus ofintense research. While the underlying cellular mechanisms areheterogeneous and can be considered marginal with respect to toxicityand disturbed homeostasis, the combined effects of these mechanisms,especially over, generate severe clinical results. For example, therapid onset and transient behavioral improvements that occur duringfever in ˜38% of children with autism may be explained by underlyingchanges in gene expression, cellular physiology, neural transmission, orsignal processing.

Furthermore, data indicate that due to chronic hypoxia, sickle celldisease (SCD) may be protective against the development of autism duringinfancy (i.e., SCD is negatively correlated with autism). Both fever andSCD may activate stress responses that improve cell survival byenhancing cellular metabolic pathways involved in homeostasis. Inductionof the stress proteome involves several interconnected pathways thatmaintain molecular integrity.

To this end, hydroxyurea (HU) is an antineoplastic agent thatsignificantly improves the quality of life of children and adults withsickle cell disease (SCD). HU has been reported also to improvecognitive function in children with SCD. (Puffer et al., 2007). HU hasbeen tested for toxicity in children ranging in age from infancy toadolescence and is FDA-approved for use in adults with SCD. Moreover, HUhas been effective in the treatment of SCD due to the increasedexpression of fetal hemoglobin. Specifically, it decreases the incidenceof strokes, improves cognitive function and is now undergoing clinicaltrials in infants with SCD.

HU is a ribonucleotide reductase and non-histone deacetylase (HDAC)inhibitor that crosses the blood-brain barrier. HU has a hormeticeffect: although toxic at high doses, at low doses, HU stimulatesexpression of the general cellular stress response and gene expression(e.g. the stress proteome). Data shown herein demonstrate that HUinduced the known cellular stress pathways that protect cells from mildmetabolic disturbances. The stress proteome response to low doses of HUincludes effects on redox regulation, DNA damage sensing and repair,molecular chaperones, fatty acid and lipid metabolism and energymetabolism. (Keefer et al., 2006). HU and related HDAC and non-HDACinhibitors are hormetic drugs that induce a general “cell-protective”response, as demonstrated in SCD as well as X-linkedadrenoleukodystrophy (ALD) (Wei et al., 2000; McGuinness et al., 2001),fragile X syndrome, and spinal muscular atrophy (SMA) (Liang et al.,2006). The cellular repair effects contrast sharply with the cellularand genetic damage reported to occur following exposure to high doses ofthe same drugs.

Thus, HU is likely to compensate for several abnormal cellular functionsin autism, e.g., mitochondrial dysfunction (Weissman et al., 2008),oxidative stress (James et al., 2009) and neuroimmune pathology (Vargaset al., 2005). Stimulation of the stress proteome by HU at low doses islikely to lead to improved clinical function in adolescents and adultswith well-characterized autism.

Administration of hydroxyurea (HU) in adolescents and adults with autismis evaluated in a study designed to ensure safety and obtain efficacydata. HU is chosen based on its effects on the stress proteome in vitroand based on clinical observations of improvements (e.g., socialresponsiveness) in persons with autism during fever. (Curran et al.,2007).

The hybrid design of the study incorporates double masking, placebocontrol, and randomization to enhance the robustness of early outcomedata. The objects of the experiment are as follows: (1) determinewhether treatment with HU administered within a specified dose range issafe (i.e., toxicity); (2) determine whether treatment with HUadministered within a specified dose range is well tolerated by autisticmale adolescents and adults (i.e., side effects and adverse events); (3)determine whether there is evidence of measurable effects on behavioralsymptoms; (4) determine whether there is evidence that treatment withinthe specified range has observable activity affecting socialresponsiveness, the most disabling core trait of autism; and (5)determine the proposed mechanism is supported by key cellular biomarkers(i.e., proof of principle).

Forty-five male adolescents (13-18 years) and adults (19-30 years) arerandomly assigned to receive either HU (n=30, at 2 month escalatingintervals: 10, 15 and 20 mg/kg/day) or placebo (n=15). Quantitativeautism traits are assessed using the ADI-R, Social Responsiveness Scale(child and adult forms), Clinical Global Impression Scale (CGI) andAutism Behavior Checklist (ABC). Prior to each dose escalation and atthe end of the study, the ADI-Current State Algorithm, SRS, CGI, and ABCare performed. Medical exams and laboratory monitoring are performed atregular intervals to observe for unanticipated signs of toxicity, andstopping rules applied by safety monitors. Statistical analyses of dataare used to describe the study sample, using individual trends for eachsubject. As the placebo effect in drug studies of autism is frequentlymarked, the duration of treatment and rigorous blinding of the presentstudy is sufficient to overcome this effect.

Data demonstrate that HU and related drugs activate cellular repair incultures from a variety of genetic disorders, such as fragile Xsyndrome, ALD and SMA, as well as SCD, which are etiologically andclinically diverse.

vi) Effect of Hydroxyurea on Nitric Oxide in Autism

Autism is a clinically heterogeneous disorder and the genetic andenvironmental factors underlying its various forms are the focus ofintense research. While the underlying cellular mechanisms areheterogeneous and can be considered marginal with respect to toxicityand disturbed homeostasis, the combined effects of these mechanisms,especially over, generate severe clinical results.

Hydroxyurea (HU), an antineoplastic agent that significantly improvesthe quality of life of children and adults with sickle cell disease(SCD), has been reported also to improve cognitive function in childrenwith SCD. (Puffer et al., 2007). HU has been tested for toxicity inchildren ranging in age from infancy to adolescence and is FDA-approvedfor use in adults with SCD. HU is a ribonucleotide reductase andnon-histone deacetylase (HDAC) inhibitor that crosses the blood-brainbarrier. HU has a hormetic effect: although toxic at high doses, at lowdoses it stimulates expression of the general cellular stress responseand gene expression (e.g., the production of fetal hemoglobin in SCD).

In autism, both enhancement of under-expressed genes and nitric oxide(NO) availability to the brain occur in response to treatment with HU.HU stimulates NO production (Lou et al., 2009) and has roles inneurodevelopment in ensuring synaptic plasticity and metasynapticcolumnar organization (Gustafsson et al., 2004; Guix et al., 2005. HUsimulates the effects of fever in autism at the cellular level bystimulating mild cellular stress.

Hydroxyurea is a ribonucleotide reductase and non-histone deacetylase(HDAC) inhibitor that crosses the blood-brain barrier. HU has a hormeticeffect: although toxic at high doses, at low doses it stimulates geneexpression (e.g., the production of fetal hemoglobin in SCD). The stressproteome response to low doses of HU includes effects on redoxregulation, DNA damage and sensing and repair, molecular chaperones,fatty acid and lipid metabolism and energy metabolism. (Keefer et al.,2006). HU and related HDAC and non-HDAC inhibitors are hormetic drugsthat induce a general cell protective response, as demonstrated in SCDas well X-linked adrenoleukodystrophy. (Wei et al., 2000; McGuinness etal., 2001). The cellular repair effects contrast sharply with thecellular and genetic damage reported to occur following exposure to highdoses of the same drugs.

The effect of HU in autism is evaluated to ensure safety and efficacy(Piantadosi et al., 2005). The hybrid study design incorporates doublemasking, placebo control, and randomization to enhance the robustness ofearly outcome data. The objectives of the study are as follows: (1)whether HU treatment administered within a specified dose range is safe(i.e., toxicity); (2) whether HU treatment administered within aspecified dose range is well tolerated by autistic male adolescents andadults (i.e., side effects and adverse events); (3) whether there isevidence of measurable effects on behavioral symptoms; (4) whether thereis evidence that treatment within the specified range has observableactivity affecting social responsiveness, which is a core trait ofautism, and (5) whether the levels of cellular biomarkers support theproposed mechanism (i.e., proof of principle).

Male adolescent (n=15, ages 13-18 years) and adults (n=30, ages 19-30)are randomly assigned to receive either HU (at 2 month escalatingintervals: 10, 15 and 30 mg/day) or placebo. Quantitative autism traitsare assessed using the ADI-R, Social Responsiveness Scale (child andadult forms) (Constantino et al., 2005), Clinical Global ImpressionsScale (CGI), and Autism Behavior Checklist (ABC). Prior to each doseescalation and at the end of the study, the ADI-Current State Algorithm,SRS, CGI, and ABC are performed. Medical exams and laboratory monitoringare performed at regular intervals to observe for signs of toxicity, andstoppage rules applied by safety monitors. Statistical analyses of dataare used to describe the study sample, using individual trends for eachsubject. As the placebo effect in drug studies of autism is frequentlymarked, the duration of treatment and rigorous blinding of the presentstudy is sufficient to overcome this effect.

Stimulation of the stress proteome and increased NO production by HU atlow doses lead to improved clinical function in adolescents and adultswith well characterized autism. HU compensates for several abnormalcellular functions in autism, e.g., mitochondrial dysfunction (Weissmanet al., 2008), oxidative stress (James et al., 2009), and neuroimmunepathology (Vargas et al., 2005).

The data demonstrate that HU and related drugs activate cellular repairin cultures from a variety of rare genetic disorders and SCD, which areetiologically and clinically diverse.

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What is claimed is:
 1. A method of treating autism or one or more autismspectrum disorders, comprising, administering to a subject diagnosedwith autism or one or more autism spectrum disorders an effective amountof a composition comprising hydroxyurea; and allowing the cells of thesubject to return to homeostasis substantially equivalent to the statethat existed in the cells prior to administering the compound.
 2. Themethod of claim 1, wherein in at least one cell of the subject, thestress proteome is stimulated.
 3. The method of claim 1, wherein in atleast one cell of the subject, increased nitrous oxide production isdetected.
 4. The method of claim 1, wherein in at least one cell of thesubject stress-sensing organelles are increased from an amount prior tothe administration of the composition.
 5. The method of claim 4, whereinthe stress-sensing organelle is a mitochondrion.
 6. The method of claim4, wherein the stress-sensing organelle is a peroxisome.
 7. The methodof claim 1, wherein the general cellular stress response comprises atleast one of mitochondrial biogenesis, peroxisome proliferation,activation of the stress proteome, transcription and/or translation ofgenes and proteins encoded by genes comprising heat shock and unfoldedprotein, genes for autophagic responses, genes for antioxidantresponses, and genes for the c-jun-N-terminal kinase pathway.
 8. Themethod of claim 7, wherein genes for heat shock proteins comprise heatshock protein 40, 70, and/or 90 family members; unfolded protein genescomprise glucose regulated protein 78 (BIP), PERK, inositol requiring 1(IRE1) and activating transcription factor 6; autophagic response genescomprise beclin-1 (BCN1), autophagy protein 5 (ATG5) andmicrotubule-associated protein 1 light chain 3 (LC3 or APG8); andantioxidant response genes comprises expression of nuclear factorerythroid 2-like 2 (NFE2L2), heme oxygenase 1 (HMOX1) and superoxidedismutase 2 (SOD2).
 9. The method of claim 1, wherein the at least onecell of the subject is located in the brain of the subject.
 10. Themethod of claim 1, wherein the at least one cell of the subject is notlocated in the brain of the subject.
 11. The method of claim 1, whereinthe composition comprises at least one of a histone deacetylaseinhibitor, Class I histone deacetylase inhibitor, or a Class II histonedeacetylase inhibitor.
 12. The method of claim 1, wherein thecomposition is a pharmaceutical composition, a medical food, a food, adrink, or a dietary supplement.
 13. A method of treating autism or oneor more autism spectrum disorders, comprising, administering to asubject diagnosed with autism or one or more autism spectrum disordersan effective amount of a pharmaceutical composition comprisinghydroxyurea.
 14. A method for determining effectiveness of a compound intreating autism or one or more autism related disorders, comprising,contacting a first cell with an effective amount of a test compound,comparing the response of the first cell to the response of a second,optionally identical, cell that was contacted with hydroxyurea, anddetermining whether or not the test compound induces a general cellularstress response in cells.
 15. The method of claim 14, wherein the cellis a normal human fibroblast, a XALD fibroblast, or a K562 cell.
 16. Themethod of claim 14, wherein the contacting occurs in vitro or in vivo.17. A method of treating autism or one or more autism spectrumdisorders, comprising, administering to a subject diagnosed with autismor one or more autism spectrum disorders an effective amount of acomposition comprising hydroxy that induces a general cellular stressresponse in at least one cell of the subject; and allowing the cells ofthe subject to return to homeostasis substantially equivalent to thestate that existed in the cells prior to administering the compound.